Products and processes for multiplex nucleic acid identification

ABSTRACT

Provided herein are products and processes for detecting the presence or absence of multiple target nucleic acids. Certain methods include amplifying the target nucleic acids, or portion thereof; extending oligonucleotides that specifically hybridize to the amplicons, where the extended oligonucleotides include a capture agent; capturing the extended oligonucleotides to a solid phase via the capture agent; releasing the extended oligonucleotide by competition with a competitor; detecting the extended oligonucleotide, and thereby determining the presence or absence of each target nucleic acid by the presence or absence of the extended oligonucleotide.

RELATED PATENT APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 16/793,732 filed on Feb. 18, 2020, entitled PRODUCTS ANDPROCESSES FOR MULTIPLEX NUCLEIC ACID IDENTIFICATION, naming ChristianeHonisch, Dirk J. Van Den Boom, Michael Mosko, and Anders Nygren asinventors, and designated by Attorney Docket No. AGB-602000N2, which isa continuation of U.S. patent application Ser. No. 13/551,486 filed onJul. 17, 2012, now issued U.S. Pat. No. 10,604,791, entitled PRODUCTSAND PROCESSES FOR MULTIPLEX NUCLEIC ACID IDENTIFICATION, namingChristiane Honisch, Dirk J. Van Den Boom, Michael Mosko, and AndersNygren as inventors, and designated by Attorney Docket No.AGB-6020-CT2t, which is a continuation application of internationalpatent application no. PCT/US2012/038710 filed on May 18, 2012, entitledPRODUCTS AND PROCESSES FOR MULTIPLEX NUCLEIC ACID IDENTIFICATION, namingChristiane Honisch, Dirk Johannes Van Den Boom, Michael Mosko, andAnders Nygren as inventors and designated by Attorney Docket No.AGB-6020-PC2, which claims the benefit of U.S. Provisional PatentApplication No. 61/488,082 filed on May 19, 2011, entitled PRODUCTS ANDPROCESSES FOR MULTIPLEX NUCLEIC ACID IDENTIFICATION, naming ChristianeHonisch, Dirk Johannes Van Den Boom, and Michael Mosko as inventors, anddesignated by Attorney Docket No. AGB-6020-PV2, and this patentapplication is related to U.S. patent application Ser. No. 13/126,684filed on Oct. 27, 2009, entitled PRODUCTS AND PROCESSES FOR MULTIPLEXNUCLEIC ACID IDENTIFICATION, naming Dirk Johannes Van Den Boom,Christiane Honisch, Andrew Timms and Smita Chitnis as inventors, anddesignated by Attorney Docket No. AGB-6020-US, which is a national phaseapplication of international patent application numberPCT/US2009/062239, filed on Oct. 27, 2009, entitled PRODUCTS ANDPROCESSES FOR MULTIPLEX NUCLEIC ACID IDENTIFICATION, naming DirkJohannes Van Den Boom, Christiane Honisch, Andrew Timms and SmitaChitnis as applicants and inventors, and designated by Attorney DocketNo. AGB-6020-PC, which claims the benefit of U.S. Provisional PatentApplication No. 61/109,885 filed on Oct. 30, 2008, entitled PRODUCTS ANDPROCESSES FOR MULTIPLEX NUCLEIC ACID IDENTIFICATION, naming DirkJohannes Van Den Boom, Christiane Honisch, Andrew Timms and SmitaChitnis as inventors, and designated by Attorney Docket No. AGB-6020-PV.The entire content of the foregoing patent applications hereby isincorporated by reference, including all text, tables and drawings.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in XML format via PatentCenter and is hereby incorporated byreference in its entirety. Said XML copy, created on Jun. 21, 2023, isnamed AGB-602000N3 SL.xml and is 526,632 bytes in size.

FIELD

The technology relates in part to nucleic acid identification proceduresin which multiple target nucleic acids can be detected in one procedure.The technology also in part relates to identification of nucleic acidmodifications.

BACKGROUND

The detection of specific nucleic acids is an important tool fordiagnostic medicine and molecular biology research. Nucleic acid assayscurrently play roles in identifying infectious organisms such asbacteria and viruses, in probing the expression of normal genes andidentifying mutant genes such as oncogenes, in typing tissue forcompatibility preceding tissue transplantation, in matching tissue orblood samples for forensic medicine, and for exploring homology amonggenes from different species, for example.

SUMMARY

Provided in some embodiments is a method for determining the presence orabsence of a plurality of target nucleic acids in a composition, whichincludes: (a) preparing amplicons of the target nucleic acids byamplifying the target nucleic acids, or portions thereof, underamplification conditions; (b) contacting the amplicons in solution witha set of oligonucleotides under hybridization conditions, where eacholigonucleotide in the set includes a hybridization sequence capable ofspecifically hybridizing to one amplicon under the hybridizationconditions when the amplicon is present in the solution; (c) generatingextended oligonucleotides that include a capture agent by extendingoligonucleotides hybridized to the amplicons by one or more nucleotides,wherein one of the one of more nucleotides is a terminating nucleotideand one or more of the nucleotides added to the oligonucleotidesincludes the capture agent; (d) contacting the extended oligonucleotideswith a solid phase under conditions in which the capture agent interactswith the solid phase; (e) releasing the extended oligonucleotides thathave interacted with the solid phase by competition with a competitor;and (f) detecting the extended oligonucleotides released in (e); wherebythe presence or absence of each target nucleic acid is determined by thepresence or absence of the corresponding extended oligonucleotide. Incertain embodiments, (i) the mass of one oligonucleotide speciesdetectably differs from the masses of the other oligonucleotide speciesin the set; and (ii) each oligonucleotide species specificallycorresponds to a specific amplicon and thereby specifically correspondsto a specific target nucleic acid. In some embodiments, (i) eacholigonucleotide in the set includes a mass distinguishable tag located5′ of the hybridization sequence, (ii) the mass of the massdistinguishable tag of one oligonucleotide detectably differs from themasses of mass distinguishable tags of the other oligonucleotides in theset; and (iii) each mass distinguishable tag specifically corresponds toa specific amplicon and thereby specifically corresponds to a specifictarget nucleic acid, the mass of the mass distinguishable tag isdetected by mass spectrometry, and the presence or absence of eachtarget nucleic acid is determined by the presence or absence of thecorresponding mass distinguishable tag. In some embodiments, detectingthe mass distinguishable tag detects the extended oligonucleotide. Incertain embodiments, the extended oligonucleotides released in (e), orthe mass distinguishable tags associated or cleaved from the releasedextended oligonucleotides, are detected by mass spectrometry.

Provided also in certain embodiments is a method for determining thepresence or absence of a plurality of target nucleic acids in acomposition, which includes: (a) preparing amplicons of the targetnucleic acids by amplifying the target nucleic acids, or portionsthereof, under amplification conditions; (b) contacting the amplicons insolution with a set of oligonucleotides under hybridization conditions,where: (i) each oligonucleotide in the set includes a hybridizationsequence capable of specifically hybridizing to one amplicon under thehybridization conditions when the amplicon is present in the solution,(ii) each oligonucleotide in the set includes a mass distinguishable taglocated 5′ of the hybridization sequence, (iii) the mass of the massdistinguishable tag of one oligonucleotide detectably differs from themasses of mass distinguishable tags of the other oligonucleotides in theset; and (iv) each mass distinguishable tag specifically corresponds toa specific amplicon and thereby specifically corresponds to a specifictarget nucleic acid; (c) generating extended oligonucleotides thatinclude a capture agent by extending oligonucleotides hybridized to theamplicons by one or more nucleotides, wherein one of the one of morenucleotides is a terminating nucleotide and one or more of thenucleotides added to the oligonucleotides includes the capture agent;(d) contacting the extended oligonucleotides with a solid phase underconditions in which the capture agent interacts with the solid phase;(e) releasing the extended oligonucleotides that have interacted withthe solid phase by competition with a competitor; and (f) detecting themass distinguishable tags released in (e); whereby the presence orabsence of each target nucleic acid is determined by the presence orabsence of the corresponding mass distinguishable tag. In certainembodiments, the extended oligonucleotides released in (e), or the massdistinguishable tags associated or cleaved from the released extendedoligonucleotides, are detected by mass spectrometry.

In some embodiments, the mass distinguishable tag is not cleaved andreleased from the extended oligonucleotide, and in certain embodiments,the mass distinguishable tag is cleaved and released from the extendedoligonucleotide. In some embodiments, the mass distinguishable tag isthe extended oligonucleotide. In certain embodiments, the extension in(c) is performed once yielding one extended oligonucleotide. In someembodiments, the extension in (c) is performed multiple times (e.g.,under amplification conditions) yielding multiple copies of the extendedoligonucleotide. In certain embodiments, a solution containing amplicons(e.g., amplicons produced in (a)) is treated with an agent that removesterminal phosphates from any nucleotides not incorporated into theamplicons. The terminal phosphate sometimes is removed by contacting theamplicons with a phosphatase, and in certain embodiments the phosphataseis alkaline phosphatase (e.g., shrimp alkaline phosphatase).

Also provided in some embodiments is a method for determining thepresence or absence of a plurality of target nucleic acids in acomposition, which comprises (a) contacting target nucleic acids insolution with a set of oligonucleotides under hybridization conditions,where (i) each oligonucleotide in the set comprises a hybridizationsequence capable of specifically hybridizing to one target nucleic acidspecies under the hybridization conditions when the target nucleic acidspecies is present in the solution; (b) generating extendedoligonucleotides that comprise a capture agent by extendingoligonucleotides hybridized to the amplicons by one or more nucleotidesunder amplification conditions, wherein one of the one of morenucleotides is a terminating nucleotide and one or more of thenucleotides added to the oligonucleotides comprises the capture agent;(c) contacting the extended oligonucleotides with a solid phase underconditions in which the capture agent interacts with the solid phase;(d) releasing the extended oligonucleotides that have interacted withthe solid phase by competition with a competitor; and (e) detecting theextended oligonucleotides released in (d); whereby the presence orabsence of each target nucleic acid is determined by the presence orabsence of the corresponding extended oligonucleotide. In certainembodiments, (i) the mass of one oligonucleotide species detectablydiffers from the masses of the other oligonucleotide species in the set;and (ii) each oligonucleotide species specifically corresponds to aspecific amplicon and thereby specifically corresponds to a specifictarget nucleic acid. In some embodiments, (i) each oligonucleotide inthe set includes a mass distinguishable tag located 5′ of thehybridization sequence, (ii) the mass of the mass distinguishable tag ofone oligonucleotide detectably differs from the masses of massdistinguishable tags of the other oligonucleotides in the set; and (iii)each mass distinguishable tag specifically corresponds to a specificamplicon and thereby specifically corresponds to a specific targetnucleic acid, the mass of the mass distinguishable tag is detected bymass spectrometry, and the presence or absence of each target nucleicacid is determined by the presence or absence of the corresponding massdistinguishable tag. In some embodiments, detecting the massdistinguishable tag detects the extended oligonucleotide. In someembodiments, detecting the mass distinguishable tag detects the extendedoligonucleotide. In certain embodiments, the extended oligonucleotidesreleased in (d), or the mass distinguishable tags associated or cleavedfrom the released extended oligonucleotides, are detected by massspectrometry.

Any suitable amplification procedure can be utilized in multiplexdetection assays described herein, and sometimes the following procedureis utilized in some embodiments, which comprises: (a) contacting thetarget nucleic acids with a set of first polynucleotides, where eachfirst polynucleotide comprises (1) a first complementary sequence thathybridizes to the target nucleic acid and (2) a first tag located 5′ ofthe complementary sequence; (b) preparing extended first polynucleotidesby extending the first polynucleotide; (c) joining a secondpolynucleotide to the 3′ end of the extended first polynucleotides,where the second polynucleotide comprises a second tag; (d) contactingthe product of (c) with a primer and extending the primer, where theprimer hybridizes to the first tag or second tag; and (e) amplifying theproduct of (c) with a set of primers under amplification conditions,where one primer in the set hybridizes to one of the tags and anotherprimer in the set hybridizes to the complement of the other tag. Incertain embodiments linear amplification is performed with one set ofprimers. In some embodiments, the second polynucleotide comprises anucleotide sequence that hybridizes to the target nucleic acid. Thenucleotide sequence of the first tag and the nucleotide sequence of thesecond tag are different in some embodiments, and are identical, or arecomplementary to one another, in other embodiments. In certainembodiments, the first tag and the second tag are included in each ofthe amplification products produced in (e). Such an amplificationprocess can further comprise (f) contacting the amplicons in solutionwith a set of oligonucleotides under hybridization conditions, whereeach oligonucleotide in the set comprises a hybridization sequencecapable of specifically hybridizing to one amplicon under thehybridization conditions when the amplicon is present in the solution;(g) generating extended oligonucleotides that comprise a capture agentby extending oligonucleotides hybridized to the amplicons by one or morenucleotides, where one of the one of more nucleotides is a terminatingnucleotide and one or more of the nucleotides added to theoligonucleotides comprises the capture agent; (h) contacting theextended oligonucleotides with a solid phase under conditions in whichthe capture agent interacts with the solid phase; (i) releasing theextended oligonucleotides that have interacted with the solid phase bycompetition with a competitor; and (j) detecting the released extendedoligonucleotides in (i); whereby the presence or absence of each targetnucleic acid is determined by the presence or absence of the extendedoligonucleotide. In certain embodiments, the extension in (g) isperformed once yielding one extended oligonucleotide. In someembodiments, the extension in (g) is performed multiple times (e.g.,under amplification conditions) yielding multiple copies of the extendedoligonucleotide. In certain embodiments, (i) the mass of oneoligonucleotide species detectably differs from the masses of the otheroligonucleotide species in the set; and (ii) each oligonucleotidespecies specifically corresponds to a specific amplicon and therebyspecifically corresponds to a specific target nucleic acid. In someembodiments, (i) each oligonucleotide in the set includes a massdistinguishable tag located 5′ of the hybridization sequence, (ii) themass of the mass distinguishable tag of one oligonucleotide detectablydiffers from the masses of mass distinguishable tags of the otheroligonucleotides in the set; and (iii) each mass distinguishable tagspecifically corresponds to a specific amplicon and thereby specificallycorresponds to a specific target nucleic acid, the mass of the massdistinguishable tag is detected by mass spectrometry, and the presenceor absence of each target nucleic acid is determined by the presence orabsence of the corresponding mass distinguishable tag. In someembodiments, detecting the mass distinguishable tag detects the extendedoligonucleotide. In some embodiments, detecting the mass distinguishabletag detects the extended oligonucleotide.

In some embodiments, competition with a competitor includes contactingthe solid phase with a competitor. In certain embodiments, thenucleotide that includes the capture agent is a capture agent conjugatedto a nucleotide triphosphate. In some embodiments, the nucleotidetriphosphate is a dideoxynucleotide triphosphate.

In certain embodiments, the capture agent includes a member of a bindingpair. In some embodiments, the capture agent includes biotin or a biotinanalogue, and on certain embodiments, the solid phase includes avidin orstreptavidin. In some embodiments, the capture agent includes avidin orstreptavidin, and in certain embodiments, the solid phase includesbiotin. In some embodiments, releasing the mass distinguishable tags bycompetition with a competitor is carried out under elevated temperatureconditions. In certain embodiments, the elevated temperature conditionsinclude treatment for between about 1 minute to about 10 minutes (e.g.,about 1 minute, about 2 minutes about 3 minutes, about 4 minutes, about5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9minutes or about 10 minutes) at a temperature of between about 80degrees Celsius to about 100 degrees Celsius (e.g., about 80 degreesCelsius (° C.), about 81° C., about 82° C., about 83° C., about 84° C.,about 85° C., about 86° C., about 87° C., about 88° C., about 89° C.,about 90° C., about 91° C., about 92° C., about 93° C., about 94° C.,about 95° C., about 96° C., about 97° C., about 98° C., about 99° C., or100° C.). In some embodiments, the elevated temperature conditionscomprise treatment for about 5 minutes at about 90 degrees Celsius. Incertain embodiments, (c) (e.g., generating extended oligonucleotidesthat include a capture agent by extending oligonucleotides hybridized tothe amplicons by one or more nucleotides, wherein one of the one of morenucleotides is a terminating nucleotide and one or more of thenucleotides added to the oligonucleotides includes the capture agent) iscarried out in one container and the method further comprisestransferring the released mass distinguishable tags to another containerbetween (e) and (f).

In some embodiments, the solution containing amplicons produced in (a)is treated with an agent that removes terminal phosphates from anynucleotides not incorporated into the amplicons. In certain embodiments,the terminal phosphate is removed by contacting the solution with aphosphatase. In some embodiments, the phosphatase is alkalinephosphatase, and in certain embodiments, the alkaline phosphatase isshrimp alkaline phosphatase.

In some embodiments, the terminal nucleotides in the extendedoligonucleotides comprise the capture agent. In certain embodiments, oneor more non-terminal nucleotides in the extended oligonucleotidescomprise the capture agent. In some embodiments, the hybridizationsequence is about 5 to about 200 nucleotides in length. In someembodiments, the hybridization sequence in each oligonucleotide is about5 to about 50 nucleotides in length. In certain embodiments, terminalnucleotides in the extended oligonucleotides comprise the capture agent,and sometimes one or more non-terminal nucleotides in the extendedoligonucleotides comprise the capture agent. In some embodiments, thecapture agent comprises biotin, or alternatively avidin or streptavidin,in which case the solid phase comprises avidin or streptavidin, orbiotin, respectively.

The distinguishable tag is distinguished in part by mass in certainembodiments (i.e., a mass distinguishable tag where a distinguishingfeature is mass). The distinguishable tag in some embodiments consistsof nucleotides, and sometimes the tag is about 5 nucleotides to about 50nucleotides in length. The distinguishable tag in certain embodiments isa nucleotide compomer, which sometimes is about 5 nucleotides to about35 nucleotides in length. In some embodiments, the distinguishable tagis a peptide, which sometimes is about 5 amino acids to about 100 aminoacids in length. The distinguishable tag in certain embodiments is aconcatemer of organic molecule units. In some embodiments, the tag is atrityl molecule concatemer.

In certain embodiments, the solid phase is selected from a flat surface,a silicon chip, a bead, sphere or combination of the foregoing. A solidphase sometimes is paramagnetic. In some embodiments, the solid phase isa paramagnetic bead, and in certain embodiments, the solid phaseincludes a capture agent.

In certain embodiments, the presence or absence of about 50 or moretarget nucleic acid species is detected by a method described herein. Insome embodiments, about 100 or more, 150 or more, 200 or more, 250 ormore, 300 or more, 325 or more, 350 or more, 375 or more, 400, or more,425 or more, 450 or more, 475 or more or 500 or more target nucleicacids is detected. In some embodiments, the presence, absence or amountof about 2 to 500 target nucleic acid species is detected by a methoddescribed herein (e.g., about 5, 10, 25, 50, 75, 100, 150, 200, 250,300, 350, 400, 450 target nucleic acid species). The target nucleicacids in certain embodiments are genomic DNA (e.g., human, microbial,viral, fungal or plant genomic DNA; any eukaryotic or prokaryoticnucleic acid (RNA and DNA)). In some embodiments, the oligonucleotidesare RNA or DNA.

In some embodiments, the mass spectrometry is matrix-assisted laserdesorption ionization (MALDI) mass spectrometry. In certain embodiments,the mass spectrometry is electrospray (ES) mass spectrometry. In someembodiments, the presence or absence of about 1 to about 50 or moretarget nucleic acids is detected. In certain embodiments, the massdistinguishable tag consists of nucleotides. In some embodiments, themass distinguishable tag is a nucleotide compomer. In certainembodiments, the nucleotide compomer is about 5 nucleotides to about 150nucleotides in length. In some embodiments, the target nucleic acids aregenomic DNA, and in certain embodiments, the genomic DNA is humangenomic DNA.

In some embodiments, detecting comprises an increased signal to noiseratio when releasing comprises competition with a competitor as comparedto releasing that does not comprise competition with a competitor. Insome embodiments, the detecting is with a signal to noise ratio greaterthan a signal to noise ratio for detecting after releasing withoutcompetition with a competitor. In some embodiments, a signal to noiseratio for extending only a mutant is greater than a signal to noiseratio for extending a wild type and a mutant allele. In someembodiments, the sensitivity of detecting a mutant allele is greater forextending only a mutant allele than for extending a wild type allele anda mutant allele. In some embodiments, the detecting comprises a signalto noise ratio greater than the signal to noise ratio for a method inwhich releasing does not comprise competition with a competitor.

In some embodiments provided is a method for detecting the presence,absence or amount of a plurality of genetic variants in a composition,comprising: (a) preparing a plurality of amplicons derived from aplurality of target nucleic acid species, or portions thereof, whereeach target nucleic acid species comprises a first variant and a secondvariant; (b) hybridizing the amplicons to oligonucleotide species, whereeach oligonucleotide species hybridizes to an amplicon derived from atarget nucleic acid species, thereby generating hybridizedoligonucleotide species; and (c) contacting the hybridizedoligonucleotide species with an extension composition comprising one ormore terminating nucleotides under extension conditions; where (i) atleast one of the one or more terminating nucleotides comprises a captureagent, and (ii) the hybridized oligonucleotide species that hybridize tothe first variant are extended by a terminating nucleotide and thehybridized oligonucleotide species that hybridize to the second variantare not extended by a terminating nucleotide, thereby generatingextended oligonucleotide species; (d) capturing the extendedoligonucleotide species to a solid phase that captures the captureagent; (e) releasing the extended oligonucleotide species bound to thesolid phase in (d) from the solid phase; and (f) detecting the mass ofeach extended oligonucleotide species released from the solid phase in(e) by mass spectrometry; whereby the presence, absence or amount of thegenetic variants is detected. In some embodiments, the extendedoligonucleotide species of the second variant is not detected. In someembodiments, each oligonucleotide species comprises a massdistinguishable tag located 5′ of the hybridization sequence. In someembodiments a method comprises a first variant and a second variantwhere the first variant is a lower abundance variation and the secondvariant is a higher abundance variation. In some embodiments the geneticvariants are single nucleotide polymorphism (SNP) variants, the firstvariant is a lower abundance allele and the second variant is a higherabundance allele. In some embodiments the one or more terminatingnucleotides consist of one terminating nucleotide. In some embodimentsthe one or more terminating nucleotides consist of two terminatingnucleotides. In some embodiments the one or more terminating nucleotidesconsist of three terminating nucleotides. In some embodiments the one ormore terminating nucleotides independently are selected from ddATP,ddGTP, ddCTP, ddTTP and ddUTP. In some embodiments the extensioncomposition comprises a non-terminating nucleotide. In some embodimentsthe extension composition comprises one or more extension nucleotides,which extension nucleotides comprise no capture agent. In someembodiments releasing the extended oligonucleotide species comprisescontacting the solid phase with a releasing agent. In some embodimentsthe capture agent comprises biotin or a biotin analogue, the solid phasecomprises streptavidin and the releasing agent comprises free biotin ora biotin analogue. In some embodiments, free biotin or a biotin analogueis the releasing agent. In some embodiments, free biotin or a biotinanalogue is added at a concentration of about 10 to about 100 ug/ml. Insome embodiments, free biotin or a biotin analogue is added at aconcentration of about 25 ug/ml. In some embodiments the releasing agenthas a higher affinity for the solid phase than the capture agent. Insome embodiments releasing the extended oligonucleotide speciescomprises heating from about 30° C. to about 100° C. In some embodimentsreleasing the extended oligonucleotide species comprises heating fromabout 60° C. to about 100° C. In some embodiments releasing the extendedoligonucleotide species comprises heating from 89° C. to about 100° C.In some embodiments releasing the extended oligonucleotide speciescomprises heating to about 90° C. In some embodiments, the solid phaseis washed after an extended oligonucleotide is captured. In someembodiments, the washing removes salts that produce interfering adductsin mass spectrometry analysis. In some embodiments, an extendedoligonucleotide is not contacted with a resin (e.g. an ion exchangeresin).

In some embodiments a plurality of target nucleic acid species is 20 ormore target nucleic acid species. In some embodiments a plurality oftarget nucleic acid species is 200 or more target nucleic acid species.In some embodiments a plurality of target nucleic acid species is 200 to300 target nucleic acid species.

In some embodiments the extension conditions comprise cycling 20 to 300times. In some embodiments the extension conditions comprise cycling 200to 300 times.

In some embodiments, a composition comprising a plurality of geneticvariants comprises a synthetic template. In some embodiments, acomposition comprising a plurality of genetic variants comprises asynthetic template and the amount and/or percentage of a first variantin the composition is determined wherein the synthetic templatecomprises a variant different than in the first variant and secondvariant and hybridizes to the same oligonucleotides species. In someembodiments, a plurality of amplicons comprise a synthetic template andthe amount and/or percentage of a first variant in a composition isdetermined wherein the synthetic template comprises a variant differentthan in the first variant and second variant and hybridizes to the sameoligonucleotides species.

Certain embodiments are described further in the following description,claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate certain non-limiting embodiments of thetechnology and are not necessarily drawn to scale.

FIGS. 1A-1D show amplification of a gene of interest using extension ofa gene specific primer with a universal PCR tag and a subsequent singlestrand ligation to a second universal tag followed by exonucleaseclean-up and amplification utilizing tag 1 and 2 (Approach 1).

FIG. 2 shows amplification of a gene of interest using a gene specificbiotinylated primer with a universal tag 3 that is extended on atemplate then ligated downstream to a gene specific phosphorylatedoligonucleotide tag 4 on the same strand. This product is subsequentlyamplified utilizing tag 3 and 4 (Concept2).

FIG. 3 shows the universal PCR products from both Approach 1 and 2procedures from FIGS. 1 and 2 , which can be identified using a post-PCRreaction (iPLEX Gold, SEQUENOM).

FIG. 4 shows MALDI-TOF MS spectra for genotyping of a single nucleotidepolymorphism (dbSNP #rs10063237) using an Approach 1 protocol.

FIG. 5A shows MALDI-TOF MS spectra for genotyping of rs1015731 using anApproach 2 protocol.

FIG. 5B shows MALDI-TOF MS spectra for genotyping 12 targets (e.g., a 12plex reaction) using an Approach 2 protocol.

FIG. 5C shows MALDI-TOF MS spectra for genotyping a 19 plex reactionusing an Approach 2 protocol.

FIG. 5D shows MALDI-TOF MS spectra for genotyping a 35 plex reactionusing an Approach 2 protocol.

FIG. 5E shows the genotypes acquired from MALDI-TOF MS spectra from FIG.5C (19 plex) and FIG. 5D (35 plex).

FIG. 6 shows PCR amplification and post-PCR primer extension withallele-specific extension primers containing allele-specific mass tags.

FIG. 7 shows MALDI-TOF MS spectra for 35 plex genotyping using post-PCRprimer extension with allele-specific extension primers containingallele-specific mass tags as a readout.

FIG. 8 shows MALDI-TOF MS spectra for genotyping of rs1000586 andrs10131894.

FIG. 9 shows oligonucleotides mass tags corresponding to a 70 plexassay. All oligonucleotides were diluted to a final total concentrationof 10 pmol and spotted on a 384 well chip. Values for area, peak heightand signal-to-noise ratio were collected from Typer 3.4 (SEQUENOM).

FIG. 10 shows peak areas for oligonucleotides mass tags corresponding to70 plex assay sorted by nucleotide composition. All oligonucleotideswere diluted to a final total concentration of 10 pmol and spotted on a384 well chip. Area values were collected from Typer 3.4 (SEQUENOM).

FIG. 11A shows a MALDI-TOF MS spectrum (zoomed views) of oligonucleotidetags corresponding to a 100 plex assay. FIG. 11B shows signal to noiseratios of oligonucleotide tags corresponding to a 100 plex assay. Alloligonucleotides were diluted to a final total concentration of 10, 5,2.5 or 1pmol, with 8 replicates spotted on a 384 well chip. Values forsignal-to-noise ratio were collected from Typer 3.4 (SEQUENOM). FIG. 11Cshows a MALDI-TOF MS spectrum (zoomed views) of a 100 plex assay afterPCR amplification and post-PCR primer extension with allele-specificextension primers containing allele-specific mass tags.

FIG. 12 shows extension rates for a 5 plex reaction. Comparing extensionoligonucleotides with or without a deoxyinosine, and either standardddNTPs or nucleotides containing a biotin moiety. Extension rates werecalculated by dividing the area of extended product by the total area ofthe peak (extended product and unextended oligonucleotide) in Typer 3.4(SEQUENOM). All experiments compare six DNAs.

FIG. 13 shows extension rates for 7 plex and 5 plex reactions over twoDNAs. Results compare extension by a single biotinylated ddNTP or abiotinylated dNTP and terminated by an unmodified ddNTP, and finalamounts of biotinylated dNTP or ddNTP of 210 or 420 pmol added to thereaction. Extension rates were calculated by dividing the area ofextended product by the total area (extended product and unextendedoligonucleotide) in Typer 3.4. All experiments include two replicates oftwo Centre de'Etude du Polymorphisme Humain (CEPH) DNAs, NA07019 andNA11036.

FIG. 14 shows a comparison of iPLEX Gold enzyme concentrations in anextension reaction using a 70 plex assay. All assays followed the sameprotocol except for the amount of iPLEX Gold enzyme used. Allexperiments include four replicates of the two CEPH DNAs NA06991 andNA07019. The results compare the signal-to-noise ratios of the extensionproducts from Typer 3.4 (SEQUENOM).

FIG. 15 shows a comparison of iPLEX Gold buffer concentration inextension reactions using a 70 plex assay. All assays followed the sameprotocol except for the amount of goldPLEX buffer used. All experimentsinclude four replicates of the two CEPH DNAs NA06991 and NA07019. Theresults compare the signal-to-noise ratios of the extension productsfrom Typer 3.4 (SEQUENOM).

FIGS. 16, 17, 18 and 19 show a comparison of extension oligonucleotideconcentration in extension reactions using a 70 plex assay. All assaysfollowed the same protocol except for the amount of extensionoligonucleotide used. All experiments include four replicates of the twoCEPH DNAs NA06991 and NA07019. The results compare the signal-to-noiseratios of the extension products from Typer 3.4 (SEQUENOM).

FIGS. 20 and 21 show a comparison of biotinylated ddNTP concentration inextension reactions using a 70 plex assay. All assays followed the sameprotocol except for the amount of biotinylated ddNTP used (valueindicates final amount of each biotinylated nucleotide). All experimentsinclude four replicates of the two CEPH DNAs NA06991 and NA07019. Theresults compare the signal-to-noise ratios of the extension productsfrom Typer 3.4 (SEQUENOM).

FIG. 22 shows a comparison of Solulink and Dynabeads MyOne C1 magneticstreptavidin beads for capturing the extend products. A total amount of10 pmol of each oligonucleotide corresponding to the two possiblealleles for assay rs1000586 were bound to the magnetic streptavidinbeads, in the presence of either water or varying quantities ofbiotinylated dNTPs (total 10, 100 or 500 pmol). The mass tags were thencleaved from the bound oligonucleotide with 10 U of endonuclease V. Theresults compare the area of the mass tag peaks from Typer 3.4 (SEQUENOM)and are listed in comparison with 10 pmol of an oligonucleotide whichhas a similar mass.

FIG. 23 shows analysis of the ability of endonuclease V to cleave anextension product containing a deoxyinosine nucleotide in differentlocations. The oligonucleotides were identical aside from thedeoxyinosine being 10, 15, 20 or 25 bases from the 3′ end of theoligonucleotide. After binding the oligonucleotide to the magneticstreptavidin beads, the supernatant was collected, cleaned by anucleotide removal kit (Qiagen) and then cleaved by treatment withendonuclease V (termed unbound oligonucleotide). The beads were washed,and cleaved with endonuclease V, as outlined in protocol section (termedcaptured/cleaved). The results compare the area of the peaks from Typer3.4 (SEQUENOM), and are listed as a percentage of oligonucleotidecleaved by endonuclease V without being bound to magnetic streptavidinbeads.

FIG. 24 shows a comparison of magnetic streptavidin beads andendonuclease V concentration using a 70 plex assay. All assays wereconducted using the same conditions except for the amount of magneticstreptavidin beads and endonuclease V. All experiments include fourreplicates of the CEPH DNA NA11036. The results compare thesignal-to-noise ratio from Typer 3.4.

FIGS. 25 and 26 show a comparison of magnetic streptavidin beads andendonuclease V concentration using a 70 plex assay. All assays followedthe same protocol except for the amount of magnetic streptavidin beadsand endonuclease V. All experiments include four replicates of the twoCEPH DNAs NA06991 and NA07019. The results compare the signal-to-noiseratio from Typer 3.4.

FIGS. 27A-27G show a schematic representation of a biotin competitionmethod for releasing a biotinylated amplification product of interestfrom a streptavidin coated magnetic or paramagnetic bead. In FIG. 27A, aregion of interest is PCR amplified (e.g., using uniplex or multiplexmethods) with subsequent dephosphorylation of the amplified productswith shrimp alkaline phosphatase (not shown in diagram). FIG. 27Billustrates single base extension of biotinylated dideoxynucleotidesover the residue of interest in the product amplified in panel A. FIG.27C illustrates the capture of the biotinylated extension products bystreptavidin coated magnetic beads. FIG. 27D illustrates a washing stepto remove unused reaction components followed by a capture step, tocapture the streptavidin coated magnetic beads to which the biotinylatedextension products are bound. FIG. 27E illustrates release of thebiotinylated extension products from the streptavidin coated magneticbead by competition with free biotin. FIG. 27F illustrates the purifiedbiotinylated extension products, which can be further analyzed using avariety of methods, including matrix assisted laser desorption/time offlight (MALDI-TOF) mass spectrometry (MS). For use in MALDI-TOF massspectrometry, the isolated extension products can be dispensed onto aSpectroCHIP® (SEQUENOM), for example. FIG. 27G illustrates arepresentative mass spectrum from MALDI-TOF MS analysis of abiotinylated extension product generated as described herein. SeeExample 12 for experimental details and results.

FIG. 28 is a representative mass spectrum of the mass difference ofvarious allelic variants (e.g., polymorphism) as measured by MALDI-TOFMS analysis of single base extension products generated usingbiotinylated dideoxynucleotide terminators and released from the solidsurface by biotin competition as described herein and illustrated inFIGS. 27A-27G. See Example 12 for experimental details and results. FIG.28 discloses SEQ ID NOS 337-339, respectively, in order of appearance.

FIGS. 29A-29G illustrate a flow chart showing a mechanism of a biotincompetition releasing step. FIG. 30A illustrates a flow chart showing amechanism of an inosine cleavage releasing step. Steps are the same asfor a biotin capture method through the wash step with the exceptionthat the extension oligonucleotides have a 5′ mass tag separated by aninosine residue. The mass tags are cleaved from captured productsthrough endonuclease V cleavage specific to inosine residues.

FIG. 30B illustrates a detection of cleaved mass tags on the MALDI. Themass represents a genetic variant.

FIG. 31 shows comparative results of biotin competition vs. inosinecleavage using different concentrations of 3′-biotinylatedoligonucleotide and different capture beads.

FIG. 32A and FIG. 32B show comparative results of biotin competition vs.inosine cleavage using Dynal Cl beads. The mass spectra peakrepresenting detected capture oligonucleotide and detectedquantification oligonucleotide are indicated by down arrows. FIG. 32Ashows the results of a biotin competition using Dynal C1 streptavidinbeads for capture and free-biotin for the competition. The concentrationof the biotinylated oligonucleotide and reference oligonucleotide (i.e.quantification oligonucleotide) tested was 0.031 uM. FIG. 32B shows theresults of an inosine cleavage release using Dynal C1 streptavidin beadsfor capture and endonuclease V for the release. The concentration of thebiotinylated oligonucleotide and reference oligonucleotide (i.e.quantification oligonucleotide) tested was 0.031 uM.

FIG. 33 shows an evaluation of different capture beads using acompetitor template.

FIGS. 34A-34C show mass spectrometry results from an assay using a verylow competitor template (about 30 molecules) for each bead type tested.The mass spectra peak representing the detected capture oligonucleotideand detected quantification oligonucleotide are indicated by downarrows.

FIG. 34A shows results using Dynal C1 beads. FIG. 34B shows resultsusing Solulink Beads. FIG. 34C shows results using Dynal M270 beads.

FIG. 35 shows the detection of BRAF-2 and BRAF-15 mutations usingdifferent extension compositions and demonstrates an increase in signalto noise ratio when ddNTPs corresponding to the wild type (e.g. moreabundant variant) are excluded from the extension composition.

FIG. 36 shows results of a competition assay with a 1% rare allele.

FIG. 37 illustrates a model plasmid that can be cleaved through EcoRIrestriction digest to separate the regions and more adequately reflect agenomic context.

DETAILED DESCRIPTION

Methods for determining the presence or absence of a plurality of targetnucleic acids in a composition described herein find multiple uses bythe person of ordinary skill in the art (hereafter referred to herein asthe “person of ordinary skill”). Such methods can be utilized, forexample, to:

(a) rapidly determine whether a particular target sequence (e.g. atarget sequence comprising a genetic variation) is present in a sample;(b) perform mixture analysis, e.g., identify a mixture and/or itscomposition or determine the frequency of a target sequence in a mixture(e.g., mixed communities, quasispecies); (c) detect sequence variations(e.g., mutations, single nucleotide polymorphisms) in a sample; (d)perform haplotyping determinations; (e) perform microorganism (e.g.,pathogen) typing; (f) detect the presence or absence of a microorganismtarget sequence in a sample; (g) identify disease markers; (h) detectmicrosatellites; (i) identify short tandem repeats; (j) identify anorganism or organisms; (k) detect allelic variations; (l) determineallelic frequency; (m) determine methylation patterns; (n) performepigenetic determinations; (o) re-sequence a region of a biomolecule;(p) perform analyses in human clinical research and medicine (e.g.cancer marker detection, sequence variation detection; detection ofsequence signatures favorable or unfavorable for a particular drugadministration), (q) perform HLA typing; (r) perform forensics analyses;(s) perform vaccine quality control analyses; (t) monitor treatments;(u) perform vector identity analyses; (v) perform vaccine or productionstrain quality control and (w) test strain identity (x) plants. Suchmethods also may be utilized, for example, in a variety of fields,including, without limitation, in commercial, education, medical,agriculture, environmental, disease monitoring, military defense, andforensics fields.

Target Nucleic Acids

As used herein, the term “nucleic acid” refers to an oligonucleotide orpolynucleotide, including, without limitation, natural nucleic acids(e.g., deoxyribonucleic acid (DNA), ribonucleic acid (RNA)), syntheticnucleic acids, non-natural nucleic acids (e.g., peptide nucleic acid(PNA)), unmodified nucleic acids, modified nucleic acids (e.g.,methylated DNA or RNA, labeled DNA or RNA, DNA or RNA having one or moremodified nucleotides). Reference to a nucleic acid as a “polynucleotide”refers to two or more nucleotides or nucleotide analogs linked by acovalent bond. Nucleic acids may be any type of nucleic acid suitablefor use with processes described herein. A nucleic acid in certainembodiments can be DNA (e.g., complementary DNA (cDNA), genomic DNA(gDNA), plasmids and vector DNA and the like), RNA (e.g., viral RNA,message RNA (mRNA), short inhibitory RNA (siRNA), ribosomal RNA (rRNA),tRNA and the like), and/or DNA or RNA analogs (e.g., containing baseanalogs, sugar analogs and/or a non-native backbone and the like). Anucleic acid can be in any form useful for conducting processes herein(e.g., linear, circular, supercoiled, single-stranded, double-strandedand the like). A nucleic acid may be, or may be from, a plasmid, phage,autonomously replicating sequence (ARS), centromere, artificialchromosome, chromosome, a cell, a cell nucleus or cytoplasm of a cell incertain embodiments. A nucleic acid in some embodiments is from a singlechromosome (e.g., a nucleic acid sample may be from one chromosome of asample obtained from a diploid organism). In the case of fetal nucleicacid, the nucleic acid may be from the paternal allele, the maternalallele or the maternal and paternal allele.

The term “species,” as used herein with reference to a target nucleicacid, amplicon, primer, sequence tag, polynucleotide, oroligonucleotide, refers to one nucleic acid having a nucleotide sequencethat differs by one or more nucleotides from the nucleotide sequence ofanother nucleic acid when the nucleotide sequences are aligned. Thus, afirst nucleic acid species differs from a second nucleic acid specieswhen the sequences of the two species, when aligned, differ by one ormore nucleotides (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100 or more than 100 nucleotidedifferences). In certain embodiments, the number of nucleic acidspecies, such as target nucleic acid species, amplicon species orextended oligonucleotide species, includes, but is not limited to about2 to about 10000 nucleic acid species, about 2 to about 1000 nucleicacid species, about 2 to about 500 nucleic acid species, or sometimesabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 55, 60, 65, 70, 75, 80, 80, 85, 90, 95, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000 or 10000 nucleic acid species.

In some embodiments an oligonucleotide species is hybridized to anucleic acid template (e.g. an amplicon) thereby forming a doublestranded nucleic acid and the oligonucleotide species that is hybridizedto the template is referred to herein as a hybridized oligonucleotidespecies. In some embodiments a hybridized oligonucleotide species cancomprise one or more nucleotides that are not hybridized to thetemplate. For example, a hybridized oligonucleotide species can compriseone or more mismatched nucleotides (e.g. non-complementary nucleotides)and sometimes a 5′ and/or 3′ region of nucleotides that do nothybridize. In some embodiments a hybridized oligonucleotide speciescomprises a tag (e.g. a mass distinguishable tag, a sequence tag, alight emitting tag or a radioactive tag). In some embodiments ahybridized oligonucleotide species comprises a capture agent (e.g.biotin, or any member of binding pair). In some embodiments a hybridizedoligonucleotide species comprises a terminating nucleotide.

As used herein, the term “nucleotides” refers to natural and non-naturalnucleotides. Nucleotides include, but are not limited to, naturallyoccurring nucleoside mono-, di-, and triphosphates: deoxyadenosinemono-, di- and triphosphate; deoxyguanosine mono-, di- and triphosphate;deoxythymidine mono-, di- and triphosphate; deoxycytidine mono-, di- andtriphosphate; deoxyuridine mono-, di- and triphosphate; and deoxyinosinemono-, di- and triphosphate (referred to herein as dA, dG, dT, dC, dUand dl, or A, G, T, C, U and I respectively). Nucleotides also include,but are not limited to, modified nucleotides and nucleotide analogs.Modified nucleotides and nucleotide analogs include, without limitation,deazapurine nucleotides, e.g., 7-deaza-deoxyguanosine (7-deaza-dG) and7-deaza-deoxyadenosine (7-deaza-dA) mono-, di- and triphosphates,deutero-deoxythymidine (deutero-dT) mon-, di- and triphosphates,methylated nucleotides e.g., 5-methyldeoxycytidine triphosphate, ¹³C/¹⁵Nlabeled nucleotides and deoxyinosine mono-, di- and triphosphate.Modified nucleotides, isotopically enriched nucleotides, depletednucleotides, tagged and labeled nucleotides and nucleotide analogs canbe obtained using a variety of combinations of functionality andattachment positions.

The term “composition” as used herein with reference to nucleic acidsrefers to a tangible item that includes one or more nucleic acids. Acomposition sometimes is a sample extracted from a source, but also acomposition of all samples at the source, and at times is the source ofone or more nucleic acids. A composition can comprise nucleic acids. Insome embodiments, a composition can comprise genomic DNA. In someembodiments, a composition can comprise maternal DNA, fetal DNA or amixture of maternal and fetal DNA. In some embodiments, a compositioncan comprise fragments of genomic DNA. In some embodiments a compositioncan comprise nucleic acids derived from a virus, bacteria, yeast,fungus, mammal or mixture thereof.

A nucleic acid sample may be derived from one or more sources. A samplemay be collected from an organism, mineral or geological site (e.g.,soil, rock, mineral deposit, fossil), or forensic site (e.g., crimescene, contraband or suspected contraband), for example. Thus, a sourcemay be environmental, such as geological, agricultural, combat theateror soil sources, for example. A source also may be from any type oforganism such as any plant, fungus, protistan, moneran, virus or animal,including but not limited, human, non-human, mammal, reptile, cattle,cat, dog, goat, swine, pig, monkey, ape, gorilla, bull, cow, bear,horse, sheep, poultry, mouse, rat, fish, dolphin, whale, and shark, orany animal or organism that may have a detectable nucleic acids. Sourcesalso can refer to different parts of an organism such as internal parts,external parts, living or non-living cells, tissue, fluid and the like.A sample therefore may be a “biological sample,” which refers to anymaterial obtained from a living source or formerly-living source, forexample, an animal such as a human or other mammal, a plant, abacterium, a fungus, a protist or a virus. A source can be in any form,including, without limitation, a solid material such as a tissue, cells,a cell pellet, a cell extract, or a biopsy, or a biological fluid suchas urine, blood, saliva, amniotic fluid, exudate from a region ofinfection or inflammation, or a mouth wash containing buccal cells,hair, cerebral spinal fluid and synovial fluid and organs. A sample alsomay be isolated at a different time point as compared to another sample,where each of the samples are from the same or a different source. Anucleic acid may be from a nucleic acid library, such as a cDNA or RNAlibrary, for example. A nucleic acid may be a result of nucleic acidpurification or isolation and/or amplification of nucleic acid moleculesfrom the sample. Nucleic acid provided for sequence analysis processesdescribed herein may contain nucleic acid from one sample or from two ormore samples (e.g., from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700,800, 900 or 1000 or more samples).

Nucleic acids may be treated in a variety of manners. For example, anucleic acid may be reduced in size (e.g., sheared, digested by nucleaseor restriction enzyme, de-phosphorylated, de-methylated), increased insize (e.g., phosphorylated, reacted with a methylation-specific reagent,attached to a detectable label), treated with inhibitors of nucleic acidcleavage and the like.

Nucleic acids may be provided for conducting methods described hereinwithout processing, in certain embodiments. In some embodiments, nucleicacid is provided for conducting methods described herein afterprocessing. For example, a nucleic acid may be extracted, isolated,purified or amplified from a sample. The term “isolated” as used hereinrefers to nucleic acid removed from its original environment (e.g., thenatural environment if it is naturally occurring, or a host cell ifexpressed exogenously), and thus is altered “by the hand of man” fromits original environment. An isolated nucleic acid generally is providedwith fewer non-nucleic acid components (e.g., protein, lipid) than theamount of components present in a source sample. A compositioncomprising isolated nucleic acid can be substantially isolated (e.g.,about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than99% free of non-nucleic acid components). The term “purified” as usedherein refers to nucleic acid provided that contains fewer nucleic acidspecies than in the sample source from which the nucleic acid isderived. A composition comprising nucleic acid may be substantiallypurified (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or greater than 99% free of other nucleic acid species).

Nucleic acids may be processed by a method that generates nucleic acidfragments, in certain embodiments, before providing nucleic acid for aprocess described herein. In some embodiments, nucleic acid subjected tofragmentation or cleavage may have a nominal, average or mean length ofabout 5 to about 10,000 base pairs, about 100 to about 1,000 base pairs,about 100 to about 500 base pairs, or about 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000or 10000 base pairs. Fragments can be generated by any suitable methodknown in the art, and the average, mean or nominal length of nucleicacid fragments can be controlled by selecting an appropriatefragment-generating procedure. In certain embodiments, nucleic acid of arelatively shorter length can be utilized to analyze sequences thatcontain little sequence variation and/or contain relatively largeamounts of known nucleotide sequence information. In some embodiments,nucleic acid of a relatively longer length can be utilized to analyzesequences that contain greater sequence variation and/or containrelatively small amounts of unknown nucleotide sequence information.

As used herein, the term “target nucleic acid” or “target nucleic acidspecies” refers to any nucleic acid species of interest in a sample. Atarget nucleic acid includes, without limitation, (i) a particularallele amongst two or more possible alleles, and (ii) a nucleic acidhaving, or not having, a particular mutation, nucleotide substitution,sequence variation, repeat sequence, marker or distinguishing sequence.As used herein, the term “different target nucleic acids” refers tonucleic acid species that differ by one or more features. As usedherein, the term “genetic variation” refers to nucleic acid species thatdiffer by one or more features. As used herein, the term “variant”refers to nucleic acid species that differ by one or more features.Features include, without limitation, one or more methyl groups or amethylation state, one or more phosphates, one or more acetyl groups,and one or more deletions, additions or substitutions of one or morenucleotides. Examples of one or more deletions, additions orsubstitutions of one or more nucleotides include, without limitation,the presence or absence of a particular mutation, presence or absence ofa nucleotide substitution (e.g., single nucleotide polymorphism (SNP)),presence or absence of a repeat sequence (e.g., di-, tri-, tetra-,penta-nucleotide repeat), presence or absence of a marker (e.g.,microsatellite) and presence of absence of a distinguishing sequence(e.g., a sequence that distinguishes one organism from another (e.g., asequence that distinguishes one viral strain from another viralstrain)). Different target nucleic acids may be distinguished by anyknown method, for example, by mass, binding, distinguishable tags andthe like, as described herein.

As used herein, the term “plurality of target nucleic acids” or“plurality of target nucleic acid species” refers to more than onetarget nucleic acid species. A plurality of target nucleic acids can beabout 2 to about 10000 nucleic acid species, about 2 to about 1000nucleic acid species, about 2 to about 500 nucleic acid species, orsometimes about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 80, 85, 90,95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000 or 10000 nucleic acid species, in certainembodiments. Detection or identification of nucleic acids results indetection of the target and can indicate the presence or absence of aparticular mutation, sequence variation (mutation or polymorphism) orgenetic variation (e.g. sequence variation, sequence difference orpolymorphism). Within the plurality of target nucleic acids, there maybe detection of the same or different target nucleic acids. Theplurality of target nucleic acids may also be identified quantitativelyas well as qualitatively in terms of identification. Also refer tomultiplexing below.

Amplification and Extension

A nucleic acid (e.g., a target nucleic acid) can be amplified in certainembodiments. As used herein, the term “amplifying,” and grammaticalvariants thereof, refers to a process of generating copies of a templatenucleic acid. For example, nucleic acid template may be subjected to aprocess that linearly or exponentially generates two or more nucleicacid amplicons (copies) having the same or substantially the samenucleotide sequence as the nucleotide sequence of the template, or aportion of the template. Nucleic acid amplification often is specific(e.g., amplicons have the same or substantially the same sequence), andcan be non-specific (e.g., amplicons have different sequences) incertain embodiments. Nucleic acid amplification sometimes is beneficialwhen the amount of target sequence present in a sample is low. Byamplifying the target sequences and detecting the amplicon synthesized,sensitivity of an assay can be improved, since fewer target sequencesare needed at the beginning of the assay for detection of a targetnucleic acid. A target nucleic acid sometimes is not amplified prior tohybridizing an extension oligonucleotide, in certain embodiments.

Amplification conditions are known and can be selected for a particularnucleic acid that will be amplified. Amplification conditions includecertain reagents some of which can include, without limitation,nucleotides (e.g., nucleotide triphosphates), modified nucleotides,oligonucleotides (e.g., primer oligonucleotides for polymerase-basedamplification and oligonucleotide building blocks for ligase-basedamplification), one or more salts (e.g., magnesium-containing salt), oneor more buffers, one or more polymerizing agents (e.g., ligase enzyme,polymerase enzyme), one or more nicking enzymes (e.g., an enzyme thatcleaves one strand of a double-stranded nucleic acid) and one or morenucleases (e.g., exonuclease, endonuclease, RNase). Any polymerasesuitable for amplification may be utilized, such as a polymerase with orwithout exonuclease activity, DNA polymerase and RNA polymerase, mutantforms of these enzymes, for example. Any ligase suitable for joining the5′ of one oligonucleotide to the 3′ end of another oligonucleotide canbe utilized. Amplification conditions also can include certain reactionconditions, such as isothermal or temperature cycle conditions. Methodsfor cycling temperature in an amplification process are known, such asby using a thermocycle device. The term “cycling” refers toamplification (e.g. an amplification reaction or extension reaction)utilizing a single primer or multiple primers where temperature cyclingis used. Amplification conditions also can, in some embodiments, includean emulsion agent (e.g., oil) that can be utilized to form multiplereaction compartments within which single nucleic acid molecule speciescan be amplified. Amplification is sometimes an exponential productgenerating process and sometimes is a linear product generating process.

A strand of a single-stranded nucleic acid target can be amplified andone or two strands of a double-stranded nucleic acid target can beamplified. An amplification product (amplicon), in some embodiments, isabout 10 nucleotides to about 10,000 nucleotides in length, about 10 toabout 1000 nucleotides in length, about 10 to about 500 nucleotides inlength, 10 to about 100 nucleotides in length, and sometimes about 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275,300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900 or 1000nucleotides in length.

Any suitable amplification technique and amplification conditions can beselected for a particular nucleic acid for amplification. Knownamplification processes include, without limitation, polymerase chainreaction (PCR), extension and ligation, ligation amplification (orligase chain reaction (LCR)) and amplification methods based on the useof Q-beta replicase or template-dependent polymerase (see US PatentPublication Number US20050287592). Also useful are strand displacementamplification (SDA), thermophilic SDA, nucleic acid sequence basedamplification (3SR or NASBA) and transcription-associated amplification(TAA). Reagents, apparatus and hardware for conducting amplificationprocesses are commercially available, and amplification conditions areknown and can be selected for the target nucleic acid at hand.

Polymerase-based amplification can be effected, in certain embodiments,by employing universal primers. In such processes, hybridization regionsthat hybridize to one or more universal primers are incorporated into atemplate nucleic acid. Such hybridization regions can be incorporatedinto (i) a primer that hybridizes to a target nucleic acid and isextended, and/or (ii) an oligonucleotide that is joined (e.g., ligatedusing a ligase enzyme) to a target nucleic acid or a product of (i), forexample. Amplification processes that involve universal primers canprovide an advantage of amplifying a plurality of target nucleic acidsusing only one or two amplification primers, for example.

FIG. 1 shows certain embodiments of amplification processes. In certainembodiments, only one primer is utilized for amplification (e.g., FIG.1A). In certain embodiments, two primers are utilized. Underamplification conditions at least one primer has a complementarydistinguishable tag. The gene specific extend primer has a 5′ universalPCRTag1R (e.g., FIG. 1A). It may be extended on any nucleic acid, forexample genomic DNA. The DNA or the PCR Tag1R gene specific extendprimer may be biotinylated, to facilitate clean up of the reaction. Theextended strand then is ligated by a single strand ligase to a universalphosphorylated oligonucleotide, which has a sequence that is the reversecomplement of Tag2F (universal PCR primer; FIG. 1B). To facilitatecleanup in the next step, the phosphorylated oligonucleotide can includeexonuclease resistant nucleotides at its 3′ end. During the exonucleasetreatment, all non-ligated extended strands are degraded, whereasligated products are protected and remain in the reaction (e.g., FIG.1C). A universal PCR then is performed, using Tag1R and the Tag2Fprimers, to amplify multiple targets (e.g., FIG. 1D).

FIG. 2 also shows certain embodiments of amplification processes. Insome embodiments, a method involving primer extension and ligation takesplace in the same reaction (e.g., FIG. 2A). Biotinylated PCRTag3Rgene-specific primer is an extension primer. The phosphorylatedoligonucleotide has a gene-specific sequence and binds about 40 bases(e.g., 4 to 100 or more) away from the primer extension site, to thesame strand of DNA. Thus a DNA polymerase, such as Stoffel polymerase,extends the strand, until it reaches the phosphorylated oligonucleotide.A ligase enzyme ligates the gene specific sequence of the phosphorylatedoligonucleotide to the extended strand. The 3′ end of phosphorylatedoligonucleotide has PCRTag4(RC)F as its universal tag. The biotinylatedextended strands then are bound to streptavidin beads. This approachfacilitates cleanup of the reaction (e.g., FIG. 2B). DNA, such asgenomic DNA, and the gene specific phosphorylated oligonucleotides arewashed away. A universal PCR then is performed, using Tag3R and Tag4F asprimers, to amplify different genes of interest (e.g., FIG. 2C).

Certain nucleic acids can be extended in certain embodiments. The term“extension,” and grammatical variants thereof, as used herein refers toelongating one strand of a nucleic acid. For example, an oligonucleotidethat hybridizes to a target nucleic acid or an amplicon generated from atarget nucleic acid can be extended in certain embodiments. An extensionreaction is conducted under extension conditions, and a variety of suchconditions are known and selected for a particular application.Extension conditions include certain reagents, including withoutlimitation, one or more oligonucleotides, extension nucleotides (e.g.,nucleotide triphosphates (dNTPs)), terminating nucleotides (e.g., one ormore dideoxynucleotide triphosphates (ddNTPs)), one or more salts (e.g.,magnesium-containing salt), one or more buffers (e.g., with beta-NAD,Triton X-100), and one or more polymerizing agents (e.g., DNApolymerase, RNA polymerase). Extension can be conducted under isothermalconditions or under non-isothermal conditions (e.g., thermocycledconditions), in certain embodiments. One or more nucleic acid speciescan be extended in an extension reaction, and one or more molecules ofeach nucleic acid species can be extended. A nucleic acid can beextended by one or more nucleotides, and in some embodiments, theextension product is about 10 nucleotides to about 10,000 nucleotides inlength, about 10 to about 1000 nucleotides in length, about 10 to about500 nucleotides in length, 10 to about 100 nucleotides in length, andsometimes about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 80, 85, 90, 95, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600,700, 800, 900 or 1000 nucleotides in length. Incorporation of aterminating nucleotide (e.g., ddNTP), the hybridization location, orother factors, can determine the length to which the oligonucleotide isextended. In certain embodiments, amplification and extension processesare carried out in the same detection procedure.

In some embodiments an extension reaction includes multiple temperaturecycles repeated to amplify the amount of extension product in thereaction. In some embodiments the extension reaction is cycled 2 or moretimes. In some embodiments the extension reaction is cycled 10 or moretimes. In some embodiments the extension reaction is cycled about 10,15, 20, 50, 100, 200, 300, 400, 500 or 600 or more times. In someembodiments the extension reaction is cycled 20 to times. In someembodiments the extension reaction is cycled 20 to 100 times. In someembodiments the extension reaction is cycled 20 to 300 times. In someembodiments the extension reaction is cycled 200 to 300 times.

In some embodiments a target nucleic acid (e.g. target nucleic acidspecies, oligonucleotide species, hybridized oligonucleotide species oramplicon) is extended in the presence of an extension composition wherethe target nucleic acid is extended by one nucleotide. An extensioncomposition can comprise one or more buffers, salts, enzymes (e.g.polymerases, Klenow, etc.), water, templates (e.g. DNA, RNA, amplicons,etc.), primers (e.g. oligonucleotides), nucleotide triphosphates,glycerol, macromolecular exclusion molecules and any other additivesused in the art. An extension composition can comprise terminatingnucleotides (e.g. dideoxynucleotides (e.g. ddNTPs)), non-terminating orextension nucleotides (e.g. dNTPs) or a mixture of terminatingnucleotides and non-terminating nucleotides. An extension compositionconsisting essentially of a particular terminating nucleotide orterminating nucleotides, can contain any other component of an extensioncomposition (e.g. buffers, salts, templates, primers, etc.), but doesnot contain any other terminating nucleotide or nucleotide triphosphate(e.g. dNTP) except those specified. For example an extension compositionconsisting essentially of ddTTP and ddCTP does not contain ddATP, ddGTPor any other dNTP. In some embodiments the nucleotides in an extensioncomposition are only terminating nucleotides and the target nucleic acidis extended by one nucleotide (i.e. sometimes there are no extensionnucleotides in the extension composition). In some embodiments anextension composition consists essentially of terminating nucleotides(e.g. ddNTPs). In some embodiments, a terminating nucleotide comprisesone or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20,or more) capture agents. In some embodiments, a terminating nucleotidecomprises one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 20, or more) different capture agents. In some embodiments, aterminating nucleotide comprises (e.g. is covalently bound to) one ormore (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, ormore) capture agent molecules. In some embodiments, a terminatingnucleotide comprises one capture agent molecule. In some embodiments, afirst terminating nucleotide comprises a capture agent and a secondterminating nucleotide comprises a different capture agent. In someembodiments, an extension composition comprises one or more terminatingnucleotides where each terminating nucleotide comprises a differentcapture agent. In some embodiments, an extension composition comprisesone or more terminating nucleotides where each terminating nucleotidecomprises a capture agent and the capture agent is the same. In someembodiments, an extension composition comprises a terminating nucleotideand an extension nucleotide and one or more of the nucleotides (e.g.terminating nucleotides and/or extension nucleotides) include a captureagent. In some embodiments a terminating nucleotide comprises a captureagent and the capture agent is biotin or a biotin analogue. In someembodiments, the extension composition consists essentially ofterminating nucleotides that are bound to one or more capture agents. Insome embodiments the capture agent is biotin or a biotin analogue. Abiotin analogue can be any modified biotin that effects the bindingproperties of biotin to avidin or streptavidin (e.g. 9-methylbiotin,biotin methyl ester (MEBio), desthiobiotin (DEBio), 2′-iminobiotin(IMBio), e-N-Biotinyl-L-lysine, diaminobiotin (DABio), including allbiotin analogues disclosed in Lai-Qiang et.al. (Lai-Qiang Ying and BruceP. Branchaud, Chemical Communications, 2011, 47, 8593-8595)). In someembodiments the capture agent is avidin, streptavidin or a modified formof avidin or streptavidin (e.g. nitroavidin, nitrostreptavidin,NeutrAvidin, CaptAvidin and derivatives thereof).

Any suitable extension reaction can be selected and utilized. Anextension reaction can be utilized, for example, to discriminate SNPalleles by the incorporation of deoxynucleotides and/ordideoxynucleotides to an extension oligonucleotide that hybridizes to aregion adjacent to the SNP site in a target nucleic acid. The primeroften is extended with a polymerase. In some embodiments, theoligonucleotide is extended by only one deoxynucleotide ordideoxynucleotide complementary to the SNP site. In some embodiments, anoligonucleotide may be extended by dNTP incorporation and terminated bya ddNTP, or terminated by ddNTP incorporation without dNTP extension incertain embodiments. One or more dNTP and/or ddNTP used during theextension reaction are labeled with a moiety allowing immobilization toa solid support, such as biotin, in some embodiments. Extension may becarried out using unmodified extension oligonucleotides and unmodifieddideoxynucleotides, unmodified extension oligonucleotides andbiotinylated dideoxynucleotides, extension oligonucleotides containing adeoxyinosine and unmodified dideoxynucleotides, extensionoligonucleotides containing a deoxyinosine and biotinylateddideoxynucleotides, extension by biotinylated dideoxynucleotides, orextension by biotinylated deoxynucleotide and/or unmodifieddideoxynucleotides, in some embodiments.

In some embodiments an oligonucleotide species can hybridize, underhybridization conditions, to a template (e.g. a target nucleic acidspecies) adjacent to a genetic variation or variant (e.g. the 3′ end ofthe oligonucleotide species may be located 5′ of the genetic variationsite and may be 0 to nucleotides away from the 5′ end of the geneticvariation site). Several variant may exist at a site of geneticvariation in a target nucleic acid. A genetic variant sometimes is asingle nucleotide polymorphism (SNP) or single nucleotide variant.Several single nucleotide variants may exist at a single base positionon a template target located 3′ of a hybridized oligonucleotide. Severalsingle nucleotide variants may differ by a single base located at aposition on a template target that is 3′ of a hybridized oligonucleotidespecies. In some embodiments an oligonucleotide species is extended byone nucleotide at the variant position. The oligonucleotide can beextended by any one of five terminating nucleotides (e.g. ddATP, ddUTP,ddTTP, ddGTP, ddCTP), depending on the number of variants present, insome embodiments. A target nucleic acid species and its variants, or acorresponding amplicon, can act as the template and can, in part,determine which terminating nucleotide is added to the oligonucleotidein the extension reaction. A target nucleic acid species may have two ormore variants. In some embodiments a target nucleic acid speciescomprises two variants. In some embodiments a target nucleic acidspecies comprises three variants. In some embodiments a target nucleicacid species comprises four variants. In some embodiments a targetnucleic acid species comprises no variants.

In some embodiments the amount of molecules of a target mutant variant(e.g. low abundant variant) present in an assay where the wild type(e.g. high abundance species) extension product is not generated isdetermined by the use of a synthetic template included in the extensionreaction. In some embodiments the amount of target (e.g. copy number,concentration, percentage) mutant variant (i.e. mutant extensionproducts) and/or percentage of target mutant variant in the sample isquantified by including a known amount of synthetic template in theextension reaction. In some embodiments the synthetic template canhybridize to an oligonucleotide species and contain a base substitutionat the mutant position located just 3′ of the oligonucleotide species tobe extended. In some embodiments, the base substitution is differentthan the wild type or target mutant variant (e.g. first variant, lowabundant variant, SNP). In some embodiments, the base substitutionpresent in the template is not present in the sample prior tointroduction of the template. In some embodiments a ddNTP (e.g. abiotin-ddNTP) that is complementary to the base substitution in thesynthetic template is also introduced into the reaction. In someembodiments, oligonucleotide species that hybridize to the target mutantvariant are co-amplified (e.g. co-extended) with oligonucleotide speciesthat hybridize to the synthetic template. In some embodiments, multiplereactions, that include serial dilutions of a synthetic template, areperformed to determine the amount and/or percentage of the target mutantvariant. In some embodiments, the amount and/or percentage of the targetmutant variant is determined by the amount of synthetic template thatyields equal extension product as the target mutant variant.

In some embodiments, one variant can be in greater abundance than othervariants. In some embodiments, the variant of greatest abundance isreferred to as the wild type variant. In some embodiments a targetnucleic acid species comprises a first and second variant where thesecond variant is represented in greater abundance (i.e. more templateis present). In some embodiments a target nucleic acid species comprisesa first, second and third variant where the second variant isrepresented in greater abundance over the first and third variant. Insome embodiments a target nucleic acid species comprises a first,second, third and fourth variant where the second variant is representedin greater abundance over the first, third and fourth variant. A variantthat is represented in a greater abundance generally is present at ahigher concentration or is represented by a greater number of molecules(e.g. copies) when compared to another variant. A higher concentrationcan be 2-fold or more. In some embodiments, a higher concentration is10-fold or more. In some embodiments, a higher concentration is a100-fold, a 1000-fold or 10000-fold or more. In some embodiments, asecond variant represents a wild type sequence and is present at a100-fold or higher concentration than a first variant. In someembodiments, a first variant is represented at a significantly lowerconcentration than a second variant (e.g. a wild type) where the firstvariant represents less of the target nucleic acid species. In someembodiments a first variant represents less than 30%, 20%, 15%, 10%, 8%,5%, 4%, 3%, 2%, 1%, 0.8%, 0.75%, 0.1%, 0.05%, 0.01% or less of thetarget nucleic acid species. In some embodiments a first variantrepresents between about 5% to about 0.75% of the target nucleic acidspecies. In some embodiments a first variant represents less than 30%,20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.1%, 0.05%, 0.01%or less of the total nucleic acid in a composition.

In some embodiments, a terminating nucleotide that is present (or, insome embodiments absent) in an extension composition determines whichterminating nucleotide is added to an oligonucleotide. In someembodiments, an extension composition comprises one or more terminatingnucleotides (e.g. ddNTPs). In some embodiments, an extension compositioncomprises one or more terminating nucleotides and one or morenon-terminating nucleotides (e.g. dNTPs). In some embodiments, anextension composition comprises only terminating nucleotides thatcorrespond to a specific variant (e.g. a first variant or a lessabundant variant) and therefore only allow extension of that specificvariant. In some embodiments, a terminating nucleotide that would allowextension of a second variant (e.g. a wild type or more abundantvariant) can be excluded from an extension composition therebypreventing extension of the second variant. In some embodiments, anextension composition comprises only terminating nucleotides thatcorrespond to a first and third variant and therefore only allowextension of those specific variants.

In some embodiments, an extension composition comprises only terminatingnucleotides that correspond to a first, third and fourth variant andtherefore only allow extension of the first, third and fourth variants.In some embodiments, an extension composition consists essentially ofterminating nucleotides that correspond to a first variant. In someembodiments, a method comprises contacting hybridized oligonucleotidespecies with an extension composition comprising one or more terminatingnucleotides under extension conditions where (i) at least one of the oneor more terminating nucleotides comprises a capture agent, and (ii) thehybridized oligonucleotide species that hybridize to the first variant(e.g. a less abundant variant, (e.g., less abundant SNP variant)) areextended by a terminating nucleotide and the hybridized oligonucleotidespecies that hybridize to the second variant (e.g. wild type or moreabundant variant) are not extended by a terminating nucleotide, therebygenerating extended oligonucleotide species. In some embodiments anextended oligonucleotide species of a second variant is not detected.

The term “signal to noise ratio” as used herein refers to thequantitative measurement of the quality of a signal by quantifying theratio of intensity of a signal relative to noise when using a detectionprocess (e.g. mass spectrometry). In some embodiments, an intensive peakon one spectrum has a greater signal to noise ratio than a low intensitypeak generated by the same analyte (e.g. an extended oligonucleotidespecies) on another spectrum. In some embodiments, noise is generated byextended oligonucleotide species derived from abundant variants (e.g.wild type alleles, second variants, wild type variants). In someembodiments, the signal generated from an extended oligonucleotidespecies derived from a less abundant variant (e.g. a first variant,third variant, fourth variant, mutant variant, mutant allele, SNP) isobscured by the noise generated by a more abundant extendedoligonucleotide species (e.g. a second variant, wild type variant, wildtype allele) when using mass spectrometry. The term “signal” as used inthe phrase “signal to noise ratio” herein refers to the intensity of asignal peak of an extended oligonucleotide species. In some embodiments,the term “signal” as used in the phrase “signal to noise ratio” hereingenerally refers to the intensity of a signal peak of an extendedoligonucleotide species derived from a less abundant variant (e.g. afirst variant, mutant variant, mutant allele, SNP). In some embodiments,a terminating nucleotide that would allow extension of a second variant(e.g. a wild type or more abundant variant) is excluded from anextension composition thereby preventing extension of the second variantand increasing the signal to noise ratio for a less abundant variant(e.g. a first variant, mutant variant, mutant allele, SNP). In someembodiments, a method comprises contacting hybridized oligonucleotidespecies with an extension composition comprising one or more terminatingnucleotides under extension conditions where (i) at least one of the oneor more terminating nucleotides comprises a capture agent, and (ii) thehybridized oligonucleotide species that hybridize to the first variant(e.g. a less abundant variant, (e.g., less abundant SNP variant)) areextended by a terminating nucleotide and the hybridized oligonucleotidespecies that hybridize to the second variant (e.g. wild type or moreabundant variant) are not extended by a terminating nucleotide, therebygenerating extended oligonucleotide species and increasing the signal tonoise ratio compared to a condition where both the first and secondvariants are extended. In some embodiments the detecting in (f) is witha signal to noise ratio greater than a signal to noise ratio fordetecting after releasing without competition with a competitor. In someembodiments the detecting in (f) comprises an increase in a signal tonoise ratio when the releasing step (e) comprises competition with acompetitor as compared to a releasing step that does not comprisecompetition with a competitor. In some embodiments a signal to noiseratio for extending only a mutant allele is greater than a signal tonoise ratio for extending a wild type and a mutant allele.

The term “sensitivity” as used herein refers to an amount of analytethat can be detected at a given signal-to-noise ratio when using adetection process (e.g. mass spectrometry). In some embodiments,sensitivity can be improved by decreasing the background or noise level.In some embodiments, noise is generated by extended oligonucleotidespecies derived from abundant variants (e.g. wild type alleles, secondvariants, wild type variants). In some embodiments, sensitivity isincreased when the signal generated from an extended oligonucleotidespecies derived from a more abundant extended oligonucleotide species(e.g. a second variant, wild type variant, wild type allele) is reducedor eliminated. In some embodiments, a terminating nucleotide that wouldallow extension of a second variant (e.g. a wild type or more abundantvariant) is excluded from an extension composition thereby preventingextension of the second variant and increasing the sensitivity fordetection of a less abundant variant (e.g. a first variant, mutantvariant, mutant allele, SNP). In some embodiments, a method comprisescontacting hybridized oligonucleotide species with an extensioncomposition comprising one or more terminating nucleotides underextension conditions where (i) at least one of the one or moreterminating nucleotides comprises a capture agent, and (ii) thehybridized oligonucleotide species that hybridize to the first variant(e.g. a less abundant variant, (e.g., less abundant SNP variant)) areextended by a terminating nucleotide and the hybridized oligonucleotidespecies that hybridize to the second variant (e.g. wild type or moreabundant variant) are not extended by a terminating nucleotide, therebygenerating extended oligonucleotide species and increasing thesensitivity for detection of the first variant compared to a conditionwhere both the first and second variants are extended. In someembodiments the sensitivity of detecting a mutant allele in (f) isgreater for extending only a mutant allele than for extending a wildtype and a mutant allele.

Any suitable type of nucleotides can be incorporated into anamplification product or an extension product. Nucleotides may benaturally occurring nucleotides, terminating nucleotides, ornon-naturally occurring nucleotides (e.g., nucleotide analog orderivative), in some embodiments. Certain nucleotides can comprise adetectable label and/or a member of a binding pair (e.g., the othermember of the binding pair may be linked to a solid phase), in someembodiments. A solution containing amplicons produced by anamplification process, or a solution containing extension productsproduced by an extension process, can be subjected to furtherprocessing. For example, a solution can be contacted with an agent thatremoves phosphate moieties from free nucleotides that have not beenincorporated into an amplicon or extension product. An example of suchan agent is a phosphatase (e.g., alkaline phosphatase). Amplicons andextension products also may be associated with a solid phase, may bewashed, may be contacted with an agent that removes a terminal phosphate(e.g., exposure to a phosphatase), may be contacted with an agent thatremoves a terminal nucleotide (e.g., exonuclease), may be contacted withan agent that cleaves (e.g., endonuclease, ribonuclease), and the like.

The term “oligonucleotide” as used herein refers to two or morenucleotides or nucleotide analogs linked by a covalent bond. Anoligonucleotide is of any convenient length, and in some embodiments isabout 5 to about 200 nucleotides in length, about 5 to about 150nucleotides in length, about 5 to about 100 nucleotides in length, about5 to about 75 nucleotides in length or about 5 to about 50 nucleotidesin length, and sometimes is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 80, 85,90, 95, 100, 125, 150, 175, or 200 nucleotides in length.Oligonucleotides may include deoxyribonucleic acid (DNA), ribonucleicacid (RNA), naturally occurring and/or non-naturally occurringnucleotides or combinations thereof and any chemical or enzymaticmodification thereof (e.g. methylated DNA, DNA of modified nucleotides).The length of an oligonucleotide sometimes is shorter than the length ofan amplicon or target nucleic acid, but not necessarily shorter than aprimer or polynucleotide used for amplification. An oligonucleotideoften comprises a nucleotide subsequence or a hybridization sequencethat is complementary, or substantially complementary, to an amplicon,target nucleic acid or complement thereof (e.g., about 95%, 96%, 97%,98%, 99% or greater than 99% identical to the amplicon or target nucleicacid complement when aligned). An oligonucleotide may contain anucleotide subsequence not complementary to, or not substantiallycomplementary to, an amplicon, target nucleic acid or complement thereof(e.g., at the 3′ or 5′ end of the nucleotide subsequence in the primercomplementary to or substantially complementary to the amplicon). Anoligonucleotide in certain embodiments, may contain a detectablemolecule (e.g., a tag, fluorophore, radioisotope, colormetric agent,particle, enzyme and the like) and/or a member of a binding pair, incertain embodiments (e.g., biotin/avidin, biotin/streptavidin).

The term “in solution” as used herein refers to a liquid, such as aliquid containing one or more nucleic acids, for example. Nucleic acidsand other components in solution may be dispersed throughout, and asolution often comprises water (e.g., aqueous solution). A solution maycontain any convenient number of oligonucleotide species, and thereoften are at least the same number of oligonucleotide species as thereare amplicon species or target nucleic acid species to be detected.

The term “hybridization sequence” as used herein refers to a nucleotidesequence in an oligonucleotide capable of specifically hybridizing to anamplicon, target nucleic acid or complement thereof. The hybridizationsequence is readily designed and selected and can be of a lengthsuitable for hybridizing to an amplicon, target sequence or complementthereof in solution as described herein. In some embodiments, thehybridization sequence in each oligonucleotide is about 5 to about 200nucleotides in length (e.g., about 5 to 10, about 10 to 15, about 15 to20, about 20 to 25, about 25 to 30, about 30 to 35, about 35 to 40,about 40 to 45, or about 45 to 50, about 50 to 70, about 80 to 90, about90 to 110, about 100 to 120, about 110 to 130, about 120 to 140, about130 to 150, about 140 to 160, about 150 to 170, about 160 to 180, about170 to 190, about 180 to 200 nucleotides in length).

The term “hybridization conditions” as used herein refers to conditionsunder which two nucleic acids having complementary nucleotide sequencescan interact with one another. Hybridization conditions can be highstringency, medium stringency or low stringency, and conditions forthese varying degrees of stringency are known. Hybridization conditionsoften are selected that allow for amplification and/or extensiondepending on the application of interest.

The term “specifically hybridizing to one amplicon or target nucleicacid” as used herein refers to hybridizing substantially to one ampliconspecies or target nucleic acid species and not substantially hybridizingto other amplicon species or target nucleic acid species in thesolution. Specific hybridization rules out mismatches so that, forexample, an oligonucleotide may be designed to hybridize specifically toa certain allele and only to that allele. An oligonucleotide that ishomogenously matched or complementary to an allele will specificallyhybridize to that allele, whereas if there is one or more basemismatches then no hybridization may occur.

The term “hybridization location” as used herein refers to a specificlocation on an amplicon or target nucleic acid to which another nucleicacid hybridizes. In certain embodiments, the terminus of anoligonucleotide is adjacent to or substantially adjacent to a site on anamplicon species or target nucleic acid species that has a differentsequence than another amplicon species or target nucleic acid species.The terminus of an oligonucleotide is “adjacent” to a site when thereare no nucleotides between the site and the oligonucleotide terminus.The terminus of an oligonucleotide is “substantially adjacent” to a sitewhen there are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides between thesite and the oligonucleotide terminus, in certain embodiments.

Capture Agents and Solid Phases

One or more capture agents may be utilized for the methods describedherein. There are several different types of capture agents availablefor processes described herein, including, without limitation, membersof a binding pair, for example. Examples of binding pairs, include,without limitation, (a) non-covalent binding pairs (e.g.,antibody/antigen, antibody/antibody, antibody/antibody fragment,antibody/antibody receptor, antibody/protein A or protein G,hapten/anti-hapten, biotin/avidin, biotin/streptavidin, folicacid/folate binding protein, receptor/ligand or binding portion thereof,and vitamin B12/intrinsic factor); and (b) covalent attachment pairs(e.g., sulfhydryl/maleimide, sulfhydryl/haloacetyl derivative,amine/isotriocyanate, amine/succinimidyl ester, and amine/sulfonylhalides), and the like. In some embodiments, one member of a bindingpair is in association with an extended oligonucleotide or amplificationproduct and another member in association with a solid phase. The term“in association with” as used herein refers to an interaction between atleast two units, where the two units are bound or linked to one another,for example.

The term “competitor” as used herein refers to any molecule thatcompetes with the capture agent for interaction with (e.g., binding to)the solid phase. Non-limiting examples of competitors include freecapture agent (e.g., one or the other member of a binding pair, freebiotin, free avidin/streptavidin), a competing fragment of a captureagent (e.g., a competing fragment of biotin or avidin/streptavidin), acompeting multimer of the capture agent (e.g., a biotin multimer),another competing molecule or fragment or multimer thereof, a moleculethat competes specifically for binding to the solid phase, elevated saltconditions, elevated temperature conditions, or combinations thereof. Insome embodiments, a multimer of a capture agent comprises between about2 and about 50 monomers. In some embodiments, a multimer of a captureagent comprises between about 2 and about 10 monomers. In someembodiments, a multimer of a capture agent comprises about 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 monomers. Insome embodiments, a capture agent comprising a multimer of captureagents comprises monomers that are covalently bound to each other. Insome embodiments, a capture agent comprising a multimer of captureagents comprises monomers that are not covalently bound to each other.The term “free capture agent” as used herein refers to a capture agentthat is not in association with a solid phase or extendedoligonucleotide. In some embodiments, a free capture agent can be biotinor a competing portion or fragment thereof. In certain embodiments, afree capture agent can be avidin, streptavidin, or a competing portionor fragment thereof. The term “competing portion or fragment” refers tocapture agent that is less than full size, yet still retains thefunctionality of the intact capture agent (e.g., the same, less or moreof the capture agent interaction activity with the solid support) withrespect to interaction with the other member of a binding pair (e.g., afragment or portion of biotin that still can bind to avidin orstreptavidin, a fragment or portion of avidin or streptavidin that stillcan bind to biotin). In some embodiments, a fragment of a free captureagent (e.g. a fragment of biotin), is any size that still retains thefunctionality of the intact capture agent. In some embodiments, a freecapture agent (e.g. a fragment of biotin), is any size that stillretains some of the functionality of the intact capture agent. In someembodiments, a free capture agent (e.g. a fragment of biotin), is a sizethat retains between about 30% and about 100% of the functionality ofthe intact capture agent. In some embodiments, a free capture agent(e.g. a fragment of biotin), is a size that retains about 30%, 40%, 50%,60%, 70%, 80%, 90% or 100% of the functionality of the intact captureagent.

In some embodiments, free capture agent (e.g. free biotin) is added at aconcentration from about to about 5000 ug/ml. In some embodiments, freecapture agent (e.g. free biotin) is added at a concentration of about0.1, 0.25, 0.5, 1, 2.5, 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100,200, 400, 800, 1000, 2000, 4000, 5000 ug/ml or higher. In someembodiments, free capture agent (e.g. free biotin) is added at aconcentration from about 10 to about 100 ug/ml. In some embodiments,free capture agent (e.g. free biotin) is added at a concentration ofabout 10, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ug/ml. In someembodiments, free capture agent (e.g. free biotin) is added to acomposition comprising an extended oligonucleotides species at aconcentration of about 25 ug/ml.

The term “solid support” or “solid phase” as used herein refers to aninsoluble material with which nucleic acid can be associated. Examplesof solid supports for use with processes described herein include,without limitation, arrays, beads (e.g., paramagnetic beads, magneticbeads, microbeads, nanobeads) and particles (e.g., microparticles,nanoparticles). Particles or beads having a nominal, average or meandiameter of about 1 nanometer to about 500 micrometers can be utilized,such as those having a nominal, mean or average diameter, for example,of about 10 nanometers to about 100 micrometers; about 100 nanometers toabout 100 micrometers; about 1 micrometer to about 100 micrometers;about 10 micrometers to about 50 micrometers; about 1, 5, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300,400, 500, 600, 700, 800 or 900 nanometers; or about 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85, 90, 95, 100, 200, 300,400, 500 micrometers. The term “paramagnetic” as used herein refers tomagnetism that generally occurs only in the presence of an externallyapplied magnetic field. Thus, a paramagnetic bead can be attracted to anexternally applied magnetic source, but typically does not exert its ownmagnetic field in the absence of an externally applied magnetic field.Magnetic beads comprising a ferrous core, generally exert their ownmagnetic field.

A solid support can comprise virtually any insoluble or solid material,and often a solid support composition is selected that is insoluble inwater. For example, a solid support can comprise or consist essentiallyof silica gel, glass (e.g. controlled-pore glass (CPG)), nylon,Sephadex®, Sepharose®, cellulose, a metal surface (e.g. steel, gold,silver, aluminum, silicon and copper), a magnetic material, a plasticmaterial (e.g., polyethylene, polypropylene, polyamide, polyester,polyvinylidenedifluoride (PVDF)) and the like. Beads or particles may beswellable (e.g., polymeric beads such as Wang resin) or non-swellable(e.g., CPG). Commercially available examples of beads include withoutlimitation Wang resin, Merrifield resin and Dynabeads® and SoluLink. Asolid phase (e.g. a bead) can comprise a member of a binding pair (e.g.avidin, streptavidin or derivative thereof). In some embodiments a solidphase is substantially hydrophilic. In some embodiments a solid phase(e.g. a bead) is substantially hydrophobic. In some embodiments a solidphase comprises a member of a binding pair (e.g. avidin, streptavidin orderivative thereof) and is substantially hydrophobic or substantiallyhydrophilic. In some embodiments, a solid phase comprises a member of abinding pair (e.g. avidin, streptavidin or derivative thereof) and has abinding capacity greater than about 1350 pmoles of free capture agent(e.g. free biotin) per mg solid support. In some embodiments the bindingcapacity of solid phase comprising a member of a binding pair is greaterthan 800, 900, 1000, 1100, 1200, 1250, 1300, 1350, 1400, 1450, 1500,1600, 1800, 2000 pmoles of free capture agent per mg solid support.

A solid support may be provided in a collection of solid supports. Asolid support collection comprises two or more different solid supportspecies. The term “solid support species” as used herein refers to asolid support in association with one particular solid phase nucleicacid species or a particular combination of different solid phasenucleic acid species. In certain embodiments, a solid support collectioncomprises 2 to 10,000 solid support species, 10 to 1,000 solid supportspecies or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 95, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000unique solid support species. The solid supports (e.g., beads) in thecollection of solid supports may be homogeneous (e.g., all are Wangresin beads) or heterogeneous (e.g., some are Wang resin beads and someare magnetic beads). Each solid support species in a collection of solidsupports sometimes is labeled with a specific identification tag. Anidentification tag for a particular solid support species sometimes is anucleic acid (e.g., “solid phase nucleic acid”) having a unique sequencein certain embodiments. An identification tag can be any molecule thatis detectable and distinguishable from identification tags on othersolid support species.

Solid phase nucleic acid often is single-stranded and is of any typesuitable for hybridizing nucleic acid (e.g., DNA, RNA, analogs thereof(e.g., peptide nucleic acid (PNA)), chimeras thereof (e.g., a singlestrand comprises RNA bases and DNA bases) and the like). Solid phasenucleic acid is associated with the solid support in any manner known bythe person of ordinary skill and suitable for hybridization of solidphase nucleic acid to nucleic acid. Solid phase nucleic acid may be inassociation with a solid support by a covalent linkage or a non-covalentinteraction. Non-limiting examples of non-covalent interactions includehydrophobic interactions (e.g., C18 coated solid support and tritylatednucleic acid), polar interactions, and the like. Solid phase nucleicacid may be associated with a solid support by different methodologyknown to the person of ordinary skill, which include without limitation(i) sequentially synthesizing nucleic acid directly on a solid support,and (ii) synthesizing nucleic acid, providing the nucleic acid insolution phase and linking the nucleic acid to a solid support. Solidphase nucleic acid may be linked covalently at various sites in thenucleic acid to the solid support, such as (i) at a 1′, 2′, 3′, 4′ or 5′position of a sugar moiety or (ii) a pyrimidine or purine base moiety,of a terminal or non-terminal nucleotide of the nucleic acid, forexample. The 5′ terminal nucleotide of the solid phase nucleic acid islinked to the solid support in certain embodiments.

After extended oligonucleotides are associated with a solid phase (i.e.post capture), unextended oligonucleotides and/or unwanted reactioncomponents that do not bind often are washed away or degraded. In someembodiments, a solid phase is washed after extended oligonucleotidespecies are captured. In some embodiments, a solid phase is washed afterextended oligonucleotide species are captured and prior to releasing theextended oligonucleotide species. In some embodiments, washing a solidphase removes salts. In some embodiments, washing a solid phase removessalts that produce interfering adducts in mass spectrometry. In someembodiments, washing a solid phase removes salts that interfere withmass spectrometry. In some embodiments, extended oligonucleotide speciesare contacted with an anion exchange resin after washing the solidphase. In some embodiments, extended oligonucleotide species are notcontacted with an anion exchange resin after washing the solid phase. Insome embodiments, extended oligonucleotide species are captured on asolid phase, washed one or more times, released from the solid phase andare not contacted with an anion exchange resin. Extendedoligonucleotides may be treated by one or more procedures prior todetection. For example, extended oligonucleotides may be conditionedprior to detection (e.g., homogenizing the type of cation and/or anionassociated with captured nucleic acid by ion exchange). Extendedoligonucleotides may be released from a solid phase prior to detectionin certain embodiments.

In some embodiments, an extended oligonucleotide (e.g. an extendedoligonucleotide species) is in association with a capture agentcomprising one member of a binding pair (e.g., biotin oravidin/streptavidin). In certain embodiments, an extendedoligonucleotide comprising a capture agent is captured by contacting abinding pair member with a solid phase comprising the other member ofthe binding pair (e.g., avidin/streptavidin or biotin). In certainembodiments an extended oligonucleotide is biotinylated, and the biotinmoiety with extended oligonucleotide product is captured by contactingthe biotin moiety with an avidin or streptavidin coated solid phase. Insome embodiments, an extended oligonucleotide comprises a massdistinguishable tag, and in certain embodiments, detecting the massdistinguishable tag comprises detecting the presence or absence of anextended oligonucleotide. In some embodiments, the extendedoligonucleotide is extended by one, two, three, or more nucleotides. Insome embodiments, an extended oligonucleotide bound to a solid phase isreleased from the solid phase by competition with a competitor and theextended oligonucleotide is detected. In some embodiments, an extendedoligonucleotide bound to a solid phase is released from the solid phaseby competition with a competitor and a distinguishable label in, orassociated with, the extended oligonucleotide is detected. In someembodiments, an extended oligonucleotide bound to a solid phase isreleased from the solid phase by competition with a competitor, adistinguishable label is released from the extended oligonucleotide, andthe released distinguishable label is detected.

Distinguishable Labels and Release

As used herein, the terms “distinguishable labels” and “distinguishabletags” refer to types of labels or tags that can be distinguished fromone another and used to identify the nucleic acid to which the tag isattached. A variety of types of labels and tags may be selected and usedfor multiplex methods provided herein. For example, oligonucleotides,amino acids, small organic molecules, light-emitting molecules,light-absorbing molecules, light-scattering molecules, luminescentmolecules, isotopes, enzymes and the like may be used as distinguishablelabels or tags. In certain embodiments, oligonucleotides, amino acids,and/or small molecule organic molecules of varying lengths, varyingmass-to-charge ratios, varying electrophoretic mobility (e.g., capillaryelectrophoresis mobility) and/or varying mass also can be used asdistinguishable labels or tags. Accordingly, a fluorophore,radioisotope, colormetric agent, light emitting agent, chemiluminescentagent, light scattering agent, and the like, may be used as a label. Thechoice of label may depend on the sensitivity required, ease ofconjugation with a nucleic acid, stability requirements, and availableinstrumentation. The term “distinguishable feature,” as used herein withrespect to distinguishable labels and tags, refers to any feature of onelabel or tag that can be distinguished from another label or tag (e.g.,mass and others described herein). In some embodiments, labelcomposition of the distinguishable labels and tags can be selectedand/or designed to result in optimal flight behavior in a massspectrometer and to allow labels and tags to be distinguished at highmultiplexing levels.

For methods used herein, a particular target nucleic acid species,amplicon species and/or extended oligonucleotide species often is pairedwith a distinguishable detectable label species, such that the detectionof a particular label or tag species directly identifies the presence ofa particular target nucleic acid species, amplicon species and/orextended oligonucleotide species in a particular composition.Accordingly, one distinguishable feature of a label species can be used,for example, to identify one target nucleic acid species in acomposition, as that particular distinguishable feature corresponds tothe particular target nucleic acid. Labels and tags may be attached to anucleic acid (e.g., oligonucleotide) by any known methods and in anylocation (e.g., at the 5′ of an oligonucleotide). Thus, reference toeach particular label species as “specifically corresponding” to eachparticular target nucleic acid species, as used herein, refers to onelabel species being paired with one target species. When the presence ofa label species is detected, then the presence of the target nucleicacid species associated with that label species thereby is detected, incertain embodiments.

The term “species,” as used herein with reference to a distinguishabletag or label (collectively, “label”), refers to one label that isdetectably distinguishable from another label. In certain embodiments,the number of label species, includes, but is not limited to, about 2 toabout 10000 label species, about 2 to about 500,000 label species, about2 to about 100,000, about 2 to about 50000, about 2 to about 10000, andabout 2 to about 500 label species, or sometimes about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000,30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000,400000 or 500000 label species.

The term “mass distinguishable label” as used herein refers to a labelthat is distinguished by mass as a feature. A variety of massdistinguishable labels can be selected and used, such as for example acompomer, amino acid and/or a concatemer. Different lengths and/orcompositions of nucleotide strings (e.g., nucleic acids; compomers),amino acid strings (e.g., peptides; polypeptides; compomers) and/orconcatemers can be distinguished by mass and be used as labels. Anynumber of units can be utilized in a mass distinguishable label, andupper and lower limits of such units depends in part on the mass windowand resolution of the system used to detect and distinguish such labels.Thus, the length and composition of mass distinguishable labels can beselected based in part on the mass window and resolution of the detectorused to detect and distinguish the labels.

The term “compomer” as used herein refers to the composition of a set ofmonomeric units and not the particular sequence of the monomeric units.For a nucleic acid, the term “compomer” refers to the base compositionof the nucleic acid with the monomeric units being bases. The number ofeach type of base can be denoted by B_(n) (i.e.: A_(a)C_(c)G_(g)T_(t),with A₀C₀G₀T₀ representing an “empty” compomer or a compomer containingno bases). A natural compomer is a compomer for which all componentmonomeric units (e.g., bases for nucleic acids and amino acids forpolypeptides) are greater than or equal to zero. In certain embodiments,at least one of a, c, g or t equals 1 or more (e.g., A₀C₀G₁T₀, A₁C₀G₁T₀,A₂C₁G₁T₂, A₃C₂G₁T₅). For purposes of comparing sequences to determinesequence variations, in the methods provided herein, “unnatural”compomers containing negative numbers of monomeric units can begenerated by an algorithm utilized to process data.

For polypeptides, a compomer refers to the amino acid composition of apolypeptide fragment, with the number of each type of amino acidsimilarly denoted. A compomer species can correspond to multiplesequences. For example, the compomer A₂G₃ corresponds to the sequencesAGGAG, GGGAA, AAGGG, GGAGA and others. In general, there is a uniquecompomer corresponding to a sequence, but more than one sequence cancorrespond to the same compomer. In certain embodiments, one compomerspecies is paired with (e.g., corresponds to) one target nucleic acidspecies, amplicon species and/or oligonucleotide species. Differentcompomer species have different base compositions, and distinguishablemasses, in embodiments herein (e.g., A₀C₀G₅T₀ and A₀C₅G₀T₀ are differentand mass-distinguishable compomer species). In some embodiments, a setof compomer species differ by base composition and have the same length.In certain embodiments, a set of compomer species differ by basecompositions and length.

A nucleotide compomer used as a mass distinguishable label can be of anylength for which all compomer species can be detectably distinguished,for example about 1 to 15, 5 to 20, 1 to 30, 5 to 35, 10 to 30, 15 to30, 20 to 35, 25 to 35, 30 to 40, 35 to 45, 40 to 50, or 25 to 50, orsometimes about 55, 60, 65, 70, 75, 80, 85, 90, 85 or 100, nucleotidesin length. A peptide or polypeptide compomer used as a massdistinguishable label can be of any length for which all compomerspecies can be detectably distinguished, for example about 1 to 20, 10to 30, 20 to 40, 30 to 50, 40 to 60, 50 to 70, 60 to 80, 70 to 90, or 80to 100 amino acids in length. As noted above, the limit to the number ofunits in a compomer often is limited by the mass window and resolutionof the detection method used to distinguish the compomer species.

The terms “concatemer” and “concatemer” are used herein synonymously(collectively “concatemer”) and refer to a molecule that contains two ormore units linked to one another (e.g., often linked in series;sometimes branched in certain embodiments). A concatemer sometimes is anucleic acid and/or an artificial polymer in some embodiments. Aconcatemer can include the same type of units (e.g., a homoconcatemer)in some embodiments, and sometimes a concatemer can contain differenttypes of units (e.g., a heteroconcatemer). A concatemer can contain anytype of unit(s), including nucleotide units, amino acid units, smallorganic molecule units (e.g., trityl), particular nucleotide sequenceunits, particular amino acid sequence units, and the like. Ahomoconcatemer of three particular sequence units ABC is ABCABCABC, inan embodiment. A concatemer can contain any number of units so long aseach concatemer species can be detectably distinguished from otherspecies. For example, a trityl concatemer species can contain about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 65, 70, 75, 80, 80, 85, 90, 95, 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500,600, 700, 800, 900 or 1000 trityl units, in some embodiments.

A distinguishable label can be released from a nucleic acid product(e.g., an extended oligonucleotide) in certain embodiments. The linkagebetween the distinguishable label and a nucleic acid can be of any typethat can be transcribed and cleaved, cleaved and allow for detection ofthe released label or labels (e.g., U.S. patent application publicationno. US20050287533A1, entitled “Target-Specific Compomers and Methods ofUse,” naming Ehrich et al.). Such linkages and methods for cleaving thelinkages (“cleaving conditions”) are known. In certain embodiments, alabel can be separated from other portions of a molecule to which it isattached. In some embodiments, a label (e.g., a compomer) is cleavedfrom a larger string of nucleotides (e.g., extended oligonucleotides).Non-limiting examples of linkages include linkages that can be cleavedby a nuclease (e.g., ribonuclease, endonuclease); linkages that can becleaved by a chemical; linkages that can be cleaved by physicaltreatment; and photocleavable linkers that can be cleaved by light(e.g., o-nitrobenzyl, 6-nitroveratryloxycarbonyl, 2-nitrobenzyl group).Photocleavable linkers provide an advantage when using a detectionsystem that emits light (e.g., matrix-assisted laser desorptionionization (MALDI) mass spectrometry involves the laser emission oflight), as cleavage and detection are combined and occur in a singlestep.

In certain embodiments, a label can be part of a larger unit, and can beseparated from that unit prior to detection. For example, in certainembodiments, a label is a set of contiguous nucleotides in a largernucleotide sequence, and the label is cleaved from the larger nucleotidesequence. In such embodiments, the label often is located at oneterminus of the nucleotide sequence or the nucleic acid in which itresides. In some embodiments, the label, or a precursor thereof, residesin a transcription cassette that includes a promoter sequenceoperatively linked with the precursor sequence that encodes the label.In the latter embodiments, the promoter sometimes is a RNApolymerase-recruiting promoter that generates an RNA that includes orconsists of the label. An RNA that includes a label can be cleaved torelease the label prior to detection (e.g., with an RNase).

In certain embodiments, a distinguishable label or tag is not cleavedfrom an extended oligonucleotide, and in some embodiments, thedistinguishable label or tag comprises a capture agent. In certainembodiments, detecting a distinguishable feature includes detecting thepresence or absence of an extended oligonucleotide, and in someembodiments an extended oligonucleotide includes a capture agent. Insome embodiments an extended oligonucleotide is released from a solidphase by competition with a competitor, and in certain embodimentscompetition with a competitor comprises contacting a solid phase with acompetitor. In some embodiments, releasing an extended oligonucleotidefrom a solid phase is carried out under elevated temperature conditions.In certain embodiments, the elevated temperature conditions are betweenabout 80 degrees Celsius and about 100 degrees Celsius. In someembodiments, releasing the extend oligonucleotides from the captureagent occurs under elevated temperature conditions for between about 1minute and about 10 minutes. In certain embodiments, releasing anextended oligonucleotides from a solid phase includes treatment with acompetitor (e.g., free capture agent, competing fragment of free captureagent, multimer of free capture agent, any molecule that specificallycompetes for binding to the solid phase, the like and combinationsthereof) for about 5 minutes at about 90 degrees Celsius. In someembodiments, a competitor is biotin and a solid phase comprisesavidin/streptavidin, and in certain embodiments a competitor isavidin/streptavidin and a solid phase comprises biotin.

In certain embodiments, a multiplex assay includes some oligonucleotidesthat are extended and some oligonucleotides that are not extended afterextension. In such embodiments, oligonucleotides that are not extendedoften do not bind to a solid phase, and in some embodiments,oligonucleotides that are not extended can interact with a solid phase.

In some embodiments, the ratio of competitor to capture agent attachedto a nucleotide or nucleic acid (e.g., extended oligonucleotide withincorporated capture agent (e.g., biotin)) can be 1:1. In certainembodiments, a competitor may be used in excess of capture agentassociated with an oligonucleotide, and in some embodiments, captureagent associated with an oligonucleotide may be in excess of competitor.In such embodiments, the excess sometimes is about a 5-fold excess toabout a 50,000-fold excess (e.g., about a 10-fold excess, about a100-fold excess, about a 1,000-fold excess, or about a 10,000-foldexcess).

Detection and Degree of Multiplexing

The term “detection” of a label as used herein refers to identificationof a label species. Any suitable detection device can be used todistinguish label species in a sample. Detection devices suitable fordetecting mass distinguishable labels, include, without limitation,certain mass spectrometers and gel electrophoresis devices. Examples ofmass spectrometry formats include, without limitation, Matrix-AssistedLaser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry(MS), MALDI orthogonal TOF MS (OTOF MS; two dimensional), LaserDesorption Mass Spectrometry (LDMS), Electrospray (ES) MS, Ion CyclotronResonance (ICR) MS, and Fourier Transform MS. Methods described hereinare readily applicable to mass spectrometry formats in which analyte isvolatized and ionized (“ionization MS,” e.g., MALDI-TOF MS, LDMS, ESMS,linear TOF, OTOF). Orthogonal ion extraction MALDI-TOF and axialMALDI-TOF can give rise to relatively high resolution, and thereby,relatively high levels of multiplexing. Detection devices suitable fordetecting light-emitting, light absorbing and/or light-scatteringlabels, include, without limitation, certain light detectors andphotodetectors (e.g., for fluorescence, chemiluminescence, absorbtion,and/or light scattering labels).

Methods provided herein allow for high-throughput detection or discoveryof target nucleic acid species in a plurality of target nucleic acids.Multiplexing refers to the simultaneous detection of more than onetarget nucleic acid species. General methods for performing multiplexedreactions in conjunction with mass spectrometry, are known (see, e.g.,U.S. Pat. Nos. 6,043,031, 5,547,835 and International PCT applicationNo. WO 97/37041). Multiplexing provides an advantage that a plurality oftarget nucleic acid species (e.g., some having different sequencevariations) can be identified in as few as a single mass spectrum, ascompared to having to perform a separate mass spectrometry analysis foreach individual target nucleic acid species. Methods provided hereinlend themselves to high-throughput, highly-automated processes foranalyzing sequence variations with high speed and accuracy, in someembodiments. In some embodiments, methods herein may be multiplexed athigh levels in a single reaction. Multiplexing is applicable when thegenotype at a polymorphic locus is not known, and in some embodiments,the genotype at a locus is known.

In certain embodiments, the number of target nucleic acid speciesmultiplexed include, without limitation, about 2 to 1,000 species, andsometimes about 1-3, 3-5, 5-7, 7-9, 9-11, 11-13, 13-15, 15-17, 17-19,19-21, 21-23, 23-25, 25-27, 27-29, 29-31, 31-33, 33-35, 35-37, 37-39,39-41, 41-43, 43-45, 45-47, 47-49, 49-51, 51-53, 53-55, 55-57, 57-59,59-61, 61-63, 63-65, 65-67, 67-69, 69-71, 71-73, 73-75, 75-77, 77-79,79-81, 81-83, 83-85, 85-87, 87-89, 89-91, 91-93, 93-95, 95-97, 97-101,101-103, 103-105, 105-107, 107-109, 109-111, 111-113, 113-115, 115-117,117-119, 121-123, 123-125, 125-127, 127-129, 129-131, 131-133, 133-135,135-137, 137-139, 139-141, 141-143, 143-145, 145-147, 147-149, 149-151,151-153, 153-155, 155-157, 157-159, 159-161, 161-163, 163-165, 165-167,167-169, 169-171, 171-173, 173-175, 175-177, 177-179, 179-181, 181-183,183-185, 185-187, 187-189, 189-191, 191-193, 193-195, 195-197, 197-199,199-201, 201-203, 203-205, 205-207, 207-209, 209-211, 211-213, 213-215,215-217, 217-219, 219-221, 221-223, 223-225, 225-227, 227-229, 229-231,231-233, 233-235, 235-237, 237-239, 239-241, 241-243, 243-245, 245-247,247-249, 249-251, 251-253, 253-255, 255-257, 257-259, 259-261, 261-263,263-265, 265-267, 267-269, 269-271, 271-273, 273-275, 275-277, 277-279,279-281, 281-283, 283-285, 285-287, 287-289, 289-291, 291-293, 293-295,295-297, 297-299, 299-301, 301-303, 303-305, 305-307, 307-309, 309-311,311-313, 313-315, 315-317, 317-319, 319-321, 321-323, 323-325, 325-327,327-329, 329-331, 331-333, 333-335, 335-337, 337-339, 339-341, 341-343,343-345, 345-347, 347-349, 349-351, 351-353, 353-355, 355-357, 357-359,359-361, 361-363, 363-365, 365-367, 367-369, 369-371, 371-373, 373-375,375-377, 377-379, 379-381, 381-383, 383-385, 385-387, 387-389, 389-391,391-393, 393-395, 395-397, 397-401, 401-403, 403-405, 405-407, 407-409,409-411, 411-413, 413-415, 415-417, 417-419, 419-421, 421-423, 423-425,425-427, 427-429, 429-431, 431-433, 433-435, 435-437, 437-439, 439-441,441-443, 443-445, 445-447, 447-449, 449-451, 451-453, 453-455, 455-457,457-459, 459-461, 461-463, 463-465, 465-467, 467-469, 469-471, 471-473,473-475, 475-477, 477-479, 479-481, 481-483, 483-485, 485-487, 487-489,489-491, 491-493, 493-495, 495-497, 497-501 species or more. Designmethods for achieving resolved mass spectra with multiplexed assays caninclude primer and oligonucleotide design methods and reaction designmethods. For primer and oligonucleotide design in multiplexed assays,the same general guidelines for primer design applies for uniplexedreactions, such as avoiding false priming and primer dimers, only moreprimers are involved for multiplex reactions. In addition, analyte peaksin the mass spectra for one assay are sufficiently resolved from aproduct of any assay with which that assay is multiplexed, includingpausing peaks and any other by-product peaks. Also, analyte peaksoptimally fall within a user-specified mass window, for example, withina range of 5,000-8,500 Da. Extension oligonucleotides can be designedwith respect to target sequences of a given SNP strand, in someembodiments. In such embodiments, the length often is between limitsthat can be, for example, user-specified (e.g., 17 to 24 bases or 17-26bases) and often do not contain bases that are uncertain in the targetsequence. Hybridization strength sometimes is gauged by calculating thesequence-dependent melting (or hybridization/dissociation) temperature,T_(m). A particular primer choice may be disallowed, or penalizedrelative to other choices of primers, because of its hairpin potential,false priming potential, primer-dimer potential, low complexity regions,and problematic subsequences such as GGGG. Methods and software fordesigning extension oligonucleotides (e.g., according to these criteria)are known, and include, for example, SpectroDESIGNER (SEQUENOM).

As used herein, the term “call rate” or “calling rate” refers to thenumber of calls (e.g., genotypes determined) obtained relative to thenumber of calls attempted to be obtained. In other words, for a 12-plexreaction, if 10 genotypes are ultimately determined from conductingmethods provided herein, then 10 calls have been obtained with a callrate of 10/12. Different events can lead to failure of a particularattempted assay, and lead to a call rate lower than 100%. Occasionally,in the case of a mix of dNTPs and ddNTPs for termination, inappropriateextension products can occur by pausing of a polymerase afterincorporation of one non-terminating nucleotide (i.e., dNTP), resultingin a prematurely terminated extension primer, for example. The massdifference between this falsely terminated and a correctly terminatedprimer mass extension reaction at the polymorphic site sometimes is toosmall to resolve consistently and can lead to miscalls if aninappropriate termination mix is used. The mass differences between acorrect termination and a false termination (i.e., one caused bypausing) as well between a correct termination and salt adducts as wellas a correct termination and an unspecific incorporation often ismaximized to reduce the number of miscalls.

Multiplex assay accuracy may be determined by assessing the number ofcalls obtained (e.g., correctly or accurately assessed) and/or thenumber of false positive and/or false negative events in one or moreassays. Accuracy also may be assessed by comparison with the accuracy ofcorresponding uniplex assays for each of the targets assessed in themultiplex assay. In certain embodiments, one or more methods may be usedto determine a call rate. For example, a manual method may be utilizedin conjunction with an automated or computer method for making calls,and in some embodiments, the rates for each method may be summed tocalculate an overall call rate. In certain embodiments, accuracy or callrates, when multiplexing two or more target nucleic acids (e.g., fiftyor more target nucleic acids), can be about 99% or greater, 98%, 97%,96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 87-88%, 85-86%, 83-84%, 81-82%,80%, 78-79% or 76-77%, for example. In some embodiments, a call rate foreach target species in a multiplex assay that includes about 2 to 200target species is greater than or equal to 80% or more (e.g., 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater).

In certain embodiments the error rate may be determined based on thecall rate or rate of accuracy. For example, the error rate may be thenumber of calls made in error. In some embodiments, for example, theerror rate may be 100% less the call rate or rate of accuracy. The errorrate may also be referred to as the “fail rate.” Identification of falsepositives and/or false negatives can readjust both the call and errorrates. In certain embodiments running more assays can also help inidentifying false positives and/or false negatives, thereby adjustingthe call and/or error rates. In certain embodiments, error rates, whenmultiplexing two or more target nucleic acids (e.g., fifty or moretarget nucleic acids), can be about 1% or less, 2%, 3%, 4,%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24% or 25%, for example.

Applications

Following are examples of non-limiting applications of multiplextechnology described herein.

1. Detection of Sequence Variations (e.g. Genetic Variants)

Provided are improved methods for identifying the genomic basis ofdisease and markers thereof. The sequence variation (e.g. geneticvariant) candidates that can be identified by the methods providedherein include sequences containing sequence variations that arepolymorphisms. Polymorphisms include both naturally occurring, somaticsequence variations and those arising from mutation. Polymorphismsinclude but are not limited to: sequence microvariants where one or morenucleotides in a localized region vary from individual to individual,insertions and deletions which can vary in size from one nucleotides tomillions of bases, and microsatellite or nucleotide repeats which varyby numbers of repeats. Nucleotide repeats include homogeneous repeatssuch as dinucleotide, trinucleotide, tetranucleotide or larger repeats,where the same sequence in repeated multiple times, and alsoheteronucleotide repeats where sequence motifs are found to repeat. Fora given locus the number of nucleotide repeats can vary depending on theindividual.

A polymorphic marker or site is the locus at which divergence occurs.Such a site can be as small as one base pair (an SNP). Polymorphicmarkers include, but are not limited to, restriction fragment lengthpolymorphisms (RFLPs), variable number of tandem repeats (VNTR's),hypervariable regions, minisatellites, dinucleotide repeats,trinucleotide repeats, tetranucleotide repeats and other repeatingpatterns, simple sequence repeats and insertional elements, such as Alu.Polymorphic forms also are manifested as different Mendelian alleles fora gene. Polymorphisms can be observed by differences in proteins,protein modifications, RNA expression modification, DNA and RNAmethylation, regulatory factors that alter gene expression and DNAreplication, and any other manifestation of alterations in genomicnucleic acid or organelle nucleic acids.

Furthermore, numerous genes have polymorphic regions. Since individualshave any one of several allelic variants of a polymorphic region,individuals can be identified based on the type of allelic variants ofpolymorphic regions of genes. This can be used, for example, forforensic purposes. In other situations, it is crucial to know theidentity of allelic variants that an individual has. For example,allelic differences in certain genes, for example, majorhistocompatibility complex (MHC) genes, are involved in graft rejectionor graft versus host disease in bone marrow transportation. Accordingly,it is highly desirable to develop rapid, sensitive, and accurate methodsfor determining the identity of allelic variants of polymorphic regionsof genes or genetic lesions. A method or a kit as provided herein can beused to genotype a subject by determining the identity of one or moreallelic variants of one or more polymorphic regions in one or more genesor chromosomes of the subject. Genotyping a subject using a method asprovided herein can be used for forensic or identity testing purposesand the polymorphic regions can be present in mitochondrial genes or canbe short tandem repeats.

Single nucleotide polymorphisms (SNPs) are generally biallelic systems,that is, there are two alleles that an individual can have for anyparticular marker. This means that the information content per SNPmarker is relatively low when compared to microsatellite markers, whichcan have upwards of 10 alleles. SNPs also tend to be verypopulation-specific; a marker that is polymorphic in one population cannot be very polymorphic in another. SNPs, found approximately everykilobase (see Wang et al. (1998) Science 280:1077-1082), offer thepotential for generating very high density genetic maps, which will beextremely useful for developing haplotyping systems for genes or regionsof interest, and because of the nature of SNPs, they can in fact be thepolymorphisms associated with the disease phenotypes under study. Thelow mutation rate of SNPs also makes them excellent markers for studyingcomplex genetic traits.

Much of the focus of genomics has been on the identification of SNPs,which are important for a variety of reasons. They allow indirecttesting (association of haplotypes) and direct testing (functionalvariants). They are the most abundant and stable genetic markers. Commondiseases are best explained by common genetic alterations, and thenatural variation in the human population aids in understanding disease,therapy and environmental interactions.

Sensitive detection of somatic mutations is especially valuable to thecancer research community whose interest is the identification ofgenetic determinants for the initiation and proliferation of tumors. Theinformation gained from a sensitive approach can also be used forprofiling mutations to predict patient outcomes and inform a relevanttreatment option. In some embodiments, a sensitive detection method,that can detect a genetic variant that represents less than or equal to5% of its counterpart wild type sequence, is needed. In someembodiments, a detection method that can detect less than or equal to 1%of wild type is implemented. In some embodiments, a detection methodthat can detect less than or equal to 5%, 4%, 3%, 2%, 1%, 0.8%, 0.75%,0.5%, 0.05%, or 0.01% of wild type is implemented. Additionally, withinpre-natal diagnostics, this type of method could elucidate paternallyderived mutations in utero.

In some embodiments, allelic analysis can be performed by generatingextended oligonucleotides from nucleic acid targets carrying one or moresomatic mutations (e.g., SNPs, disease markers, the like andcombinations thereof) of interest. Detecting the presence or absence ofa released, extended oligonucleotide representing an allele carrying asomatic mutation can be utilized as a rapid method of screening for thepresence or absence of a particular mutation in a target population, insome embodiments. In certain embodiments involving generating anextended oligonucleotide from a mutant allele, the extendedoligonucleotide can be detected as the appropriate mutant allele givesrise to an extended oligonucleotide product.

2. Identifying Disease Markers

Provided herein are methods for the rapid and accurate identification ofsequence variations that are genetic markers of disease, which can beused to diagnose or determine the prognosis of a disease. Diseasescharacterized by genetic markers can include, but are not limited to,atherosclerosis, obesity, diabetes, autoimmune disorders, and cancer.Diseases in all organisms have a genetic component, whether inherited orresulting from the body's response to environmental stresses, such asviruses and toxins. The ultimate goal of ongoing genomic research is touse this information to develop new ways to identify, treat andpotentially cure these diseases. The first step has been to screendisease tissue and identify genomic changes at the level of individualsamples. The identification of these “disease” markers is dependent onthe ability to detect changes in genomic markers in order to identifyerrant genes or sequence variants. Genomic markers (all genetic lociincluding single nucleotide polymorphisms (SNPs), microsatellites andother noncoding genomic regions, tandem repeats, introns and exons) canbe used for the identification of all organisms, including humans. Thesemarkers provide a way to not only identify populations but also allowstratification of populations according to their response to disease,drug treatment, resistance to environmental agents, and other factors. Adisease marker sometimes is a mutation, and can be a relatively rareallele such as, for example, a somatic mutation against the backgroundof a wild type allele (e.g., cancer tissue versus normal tissue, mutantviral type versus normal viral type (e.g. HIV)), in some embodiments. Insome embodiments the rare allele or mutation represents less than 5%,4%, 3%, 2%, 1%, 0.8%, 0.75%, 0.1%, 0.05%, or 0.01% of the wild type. Insome embodiment, the rare allele or mutation can represent less than 1%of the wild type.

3. Microbial Identification

Provided herein is a process or method for identifying genera, species,strains, clones or subtypes of microorganisms and viruses. Themicroorganism(s) and viruses are selected from a variety of organismsincluding, but not limited to, bacteria, fungi, protozoa, ciliates, andviruses. The microorganisms are not limited to a particular genus,species, strain, subtype or serotype or any other classification. Themicroorganisms and viruses can be identified by determining sequencevariations in a target microorganism sequence relative to one or morereference sequences or samples. The reference sequence(s) can beobtained from, for example, other microorganisms from the same ordifferent genus, species strain or serotype or any other classification,or from a host prokaryotic or eukaryotic organism or any mixedpopulation.

Identification and typing of pathogens (e.g., bacterial or viral) iscritical in the clinical management of infectious diseases. Preciseidentity of a microbe is used not only to differentiate a disease statefrom a healthy state, but is also fundamental to determining the sourceof the infection and its spread and whether and which antibiotics orother antimicrobial therapies are most suitable for treatment. Inaddition treatment can be monitored. Traditional methods of pathogentyping have used a variety of phenotypic features, including growthcharacteristics, color, cell or colony morphology, antibioticsusceptibility, staining, smell, serotyping, biochemical typing andreactivity with specific antibodies to identify microbes (e.g.,bacteria). All of these methods require culture of the suspectedpathogen, which suffers from a number of serious shortcomings, includinghigh material and labor costs, danger of worker exposure, falsepositives due to mishandling and false negatives due to low numbers ofviable cells or due to the fastidious culture requirements of manypathogens. In addition, culture methods require a relatively long timeto achieve diagnosis, and because of the potentially life-threateningnature of such infections, antimicrobial therapy is often started beforethe results can be obtained. Some organisms cannot be maintained inculture or exhibit prohibitively slow growth rates (e.g., up to 6-8weeks for Mycobacterium tuberculosis).

In many cases, the pathogens are present in minor amounts and/or arevery similar to the organisms that make up the normal flora, and can beindistinguishable from the innocuous strains by the methods cited above.In these cases, determination of the presence of the pathogenic straincan require the higher resolution afforded by the molecular typingmethods provided herein.

4. Detecting the Presence of Viral or Bacterial Nucleic Acid SequencesIndicative of an Infection

The methods provided herein can be used to determine the presence ofviral or bacterial nucleic acid sequences indicative of an infection byidentifying sequence variations that are present in the viral orbacterial nucleic acid sequences relative to one or more referencesequences. The reference sequence(s) can include, but are not limitedto, sequences obtained from an infectious organism, relatednon-infectious organisms, or sequences from host organisms. Viruses,bacteria, fungi and other infectious organisms contain distinct nucleicacid sequences, including sequence variants, which are different fromthe sequences contained in the host cell. A target DNA sequence can bepart of a foreign genetic sequence such as the genome of an invadingmicroorganism, including, for example, bacteria and their phages,viruses, fungi, protozoa, and the like. The processes provided hereinare particularly applicable for distinguishing between differentvariants or strains of a microorganism (e.g., pathogenic, lesspathogenic, resistant versus non-resistant and the like) in order, forexample, to choose an appropriate therapeutic intervention. Examples ofdisease-causing viruses that infect humans and animals and that can bedetected by a disclosed process include but are not limited toRetroviridae (e.g., human immunodeficiency viruses such as HIV-1 (alsoreferred to as HTLV-III, LAV or HTLV-III/LAV; Ratner et al., Nature,313:227-284 (1985); Wain Hobson et al., Cell, 40:9-17 (1985), HIV-2(Guyader et al., Nature, 328:662-669 (1987); European Patent PublicationNo. 0 269 520; Chakrabarti et al., Nature, 328:543-547 (1987); EuropeanPatent Application No. 0 655 501), and other isolates such as HIV-LP(International Publication No. WO 94/00562); Picornaviridae (e.g.,polioviruses, hepatitis A virus, (Gust et al., Intervirology, 20:1-7(1983)); enteroviruses, human coxsackie viruses, rhinoviruses,echoviruses); Calcivirdae (e.g. strains that cause gastroenteritis);Togaviridae (e.g., equine encephalitis viruses, rubella viruses);Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow feverviruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g.,vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebolaviruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g.,influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Parvoviridae (most adenoviruses); Papovaviridae(papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses);Herpesviridae (herpes simplex virus type 1 (HSV-1) and HSV-2, varicellazoster virus, cytomegalovirus, herpes viruses; Poxviridae (variolaviruses, vaccinia viruses, pox viruses); Iridoviridae (e.g., Africanswine fever virus); and unclassified viruses (e.g., the etiologicalagents of Spongiform encephalopathies, the agent of delta hepatitis(thought to be a defective satellite of hepatitis B virus), the agentsof non-A, non-B hepatitis (class 1=internally transmitted; class2=parenterally transmitted, i.e., Hepatitis C); Norwalk and relatedviruses, and astroviruses.

Examples of infectious bacteria include but are not limited toHelicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sp. (e.g. M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Salmonella, Staphylococcus aureus, Neisseriagonorrheae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus sp. (viridans group),Streptococcus faecalis, Streptococcus bovis, Streptococcus sp.(anaerobic species), Streptococcus pneumoniae, pathogenic Campylobactersp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis,Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrixrhusiopathiae, Clostridium perfringens, Clostridium tetani, Escherichiacoli, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomyces israelli and any variants including antibiotic resistancevariants

Examples of infectious fungi include but are not limited to Cryptococcusneoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomycesdermatitidis, Chlamydia trachomatis, Candida albicans. Other infectiousorganisms include protists such as Plasmodium falciparum and Toxoplasmagondii.

5. Antibiotic Profiling

Methods provided herein can improve the speed and accuracy of detectionof nucleotide changes involved in drug resistance, including antibioticresistance. Genetic loci involved in resistance to isoniazid, rifampin,streptomycin, fluoroquinolones, and ethionamide have been identified[Heym et al., Lancet 344:293 (1994) and Morris et al., J. Infect. Dis.171:954 (1995)]. A combination of isoniazid (inh) and rifampin (rif)along with pyrazinamide and ethambutol or streptomycin, is routinelyused as the first line of attack against confirmed cases of M.tuberculosis [Banerjee et al., Science 263:227 (1994)]. The increasingincidence of such resistant strains necessitates the development ofrapid assays to detect them and thereby reduce the expense and communityhealth hazards of pursuing ineffective, and possibly detrimental,treatments. The identification of some of the genetic loci involved indrug resistance has facilitated the adoption of mutation detectiontechnologies for rapid screening of nucleotide changes that result indrug resistance. In addition, the technology facilitates treatmentmonitoring and tracking or microbial population structures as well assurveillance monitoring during treatment. In addition, correlations andsurveillance monitoring of mixed populations can be performed.

6. Haplotyping

The methods provided herein can be used to detect haplotypes. In anydiploid cell, there are two haplotypes at any gene or other chromosomalsegment that contain at least one distinguishing variance. In manywell-studied genetic systems, haplotypes are more powerfully correlatedwith phenotypes than single nucleotide variations. Thus, thedetermination of haplotypes is valuable for understanding the geneticbasis of a variety of phenotypes including disease predisposition orsusceptibility, response to therapeutic interventions, and otherphenotypes of interest in medicine, animal husbandry, and agriculture.

Haplotyping procedures as provided herein permit the selection of aportion of sequence from one of an individual's two homologouschromosomes and to genotype linked SNPs on that portion of sequence. Thedirect resolution of haplotypes can yield increased information content,improving the diagnosis of any linked disease genes or identifyinglinkages associated with those diseases.

7. Microsatellites

Methods provided herein allow for rapid, unambiguous detection ofmicrosatellite sequence variations. Microsatellites (sometimes referredto as variable number of tandem repeats or VNTRs) are short tandemlyrepeated nucleotide units of one to seven or more bases, the mostprominent among them being di-, tri-, and tetranucleotide repeats.Microsatellites are present every 100,000 bp in genomic DNA (J. L. Weberand P. E. Can, Am. J. Hum. Genet. 44, 388 (1989); J. Weissenbach et al.,Nature 359, 794 (1992)). CA dinucleotide repeats, for example, make upabout of the human extra-mitochondrial genome; CT and AG repeatstogether make up about 0.2%. CG repeats are rare, most probably due tothe regulatory function of CpG islands. Microsatellites are highlypolymorphic with respect to length and widely distributed over the wholegenome with a main abundance in non-coding sequences, and their functionwithin the genome is unknown. Microsatellites can be important inforensic applications, as a population will maintain a variety ofmicrosatellites characteristic for that population and distinct fromother populations which do not interbreed.

Many changes within microsatellites can be silent, but some can lead tosignificant alterations in gene products or expression levels. Forexample, trinucleotide repeats found in the coding regions of genes areaffected in some tumors (C. T. Caskey et al., Science 256, 784 (1992)and alteration of the microsatellites can result in a geneticinstability that results in a predisposition to cancer (P. J. McKinnen,Hum. Genet. 1 75, 197 (1987); J. German et al., Clin. Genet. 35, 57(1989)).

8. Short Tandem Repeats

The methods provided herein can be used to identify short tandem repeat(STR) regions in some target sequences of the human genome relative to,for example, reference sequences in the human genome that do not containSTR regions. STR regions are polymorphic regions that are not related toany disease or condition. Many loci in the human genome contain apolymorphic short tandem repeat (STR) region. STR loci contain short,repetitive sequence elements of 3 to 7 base pairs in length. It isestimated that there are 200,000 expected trimeric and tetrameric STRs,which are present as frequently as once every 15 kb in the human genome(see, e.g., International PCT application No. WO 9213969 A1, Edwards etal., Nucl. Acids Res. 19:4791 (1991); Beckmann et al. (1992) Genomics12:627-631). Nearly half of these STR loci are polymorphic, providing arich source of genetic markers. Variation in the number of repeat unitsat a particular locus is responsible for the observed sequencevariations reminiscent of variable nucleotide tandem repeat (VNTR) loci(Nakamura et al. (1987) Science 235:1616-1622); and minisatellite loci(Jeffreys et al. (1985) Nature 314:67-73), which contain longer repeatunits, and microsatellite or dinucleotide repeat loci (Luty et al.(1991) Nucleic Acids Res. 19:4308; Litt et al. (1990) Nucleic Acids Res.18:4301; Litt et al. (1990) Nucleic Acids Res. 18:5921; Luty et al.(1990) Am. J. Hum. Genet. 46:776-783; Tautz (1989) Nucl. Acids Res.17:6463-6471; Weber et al. (1989) Am. J. Hum. Genet. 44:388-396;Beckmann et al. (1992) Genomics 12:627-631). VNTR typing is a veryestablished tool in microbial typing e.g. M. tuberculosis (MIRU typing).

Examples of STR loci include, but are not limited to, pentanucleotiderepeats in the human CD4 locus (Edwards et al., Nucl. Acids Res. 19:4791(1991)); tetranucleotide repeats in the human aromatase cytochrome P-450gene (CYP19; Polymeropoulos et al., Nucl. Acids Res. 19:195 (1991));tetranucleotide repeats in the human coagulation factor XIII A subunitgene (F13A1; Polymeropoulos et al., Nucl. Acids Res. 19:4306 (1991));tetranucleotide repeats in the F13B locus (Nishimura et al., Nucl. AcidsRes. 20:1167 (1992)); tetranucleotide repeats in the human c-les/fps,proto-oncogene (FES; Polymeropoulos et al., Nucl. Acids Res. 19:4018(1991)); tetranucleotide repeats in the LFL gene (Zuliani et al., Nucl.Acids Res. 18:4958 (1990)); trinucleotide repeat sequence variations atthe human pancreatic phospholipase A-2 gene (PLA2; Polymeropoulos etal., Nucl. Acids Res. 18:7468 (1990)); tetranucleotide repeat sequencevariations in the VWF gene (Ploos et al., Nucl. Acids Res. 18:4957(1990)); and tetranucleotide repeats in the human thyroid peroxidase(hTPO) locus (Anker et al., Hum. Mol. Genet. 1:137 (1992)).

9. Organism Identification

Polymorphic STR loci and other polymorphic regions of genes are sequencevariations that are extremely useful markers for human identification,paternity and maternity testing, genetic mapping, immigration andinheritance disputes, zygosity testing in twins, tests for inbreeding inhumans, quality control of human cultured cells, identification of humanremains, and testing of semen samples, blood stains, microbes and othermaterial in forensic medicine. Such loci also are useful markers incommercial animal breeding and pedigree analysis and in commercial plantbreeding. Traits of economic importance in plant crops and animals canbe identified through linkage analysis using polymorphic DNA markers.Efficient and accurate methods for determining the identity of such lociare provided herein.

Detecting Allelic Variation

The methods provided herein allow for high-throughput, fast and accuratedetection of allelic variants. Studies of allelic variation involve notonly detection of a specific sequence in a complex background, but alsothe discrimination between sequences with few, or single, nucleotidedifferences. One method for the detection of allele-specific variants byPCR is based upon the fact that it is difficult for Taq polymerase tosynthesize a DNA strand when there is a mismatch between the templatestrand and the 3′ end of the primer. An allele-specific variant can bedetected by the use of a primer that is perfectly matched with only oneof the possible alleles; the mismatch to the other allele acts toprevent the extension of the primer, thereby preventing theamplification of that sequence. The methods herein also are applicableto association studies, copy number variations, detection of diseasemarker and SNP sets for typing and the like.

11. Determining Allelic Frequency

The methods herein described are valuable for identifying one or moregenetic markers whose frequency changes within the population as afunction of age, ethnic group, sex or some other criteria. For example,the age-dependent distribution of ApoE genotypes is known in the art(see, e.g., Schechter et al. (1994) Nature Genetics 6:29-32). Thefrequencies of sequence variations known to be associated at some levelwith disease can also be used to detect or monitor progression of adisease state. For example, the N291S polymorphism (N291S) of theLipoprotein Lipase gene, which results in a substitution of a serine foran asparagine at amino acid codon 291, leads to reduced levels of highdensity lipoprotein cholesterol (HDL-C) that is associated with anincreased risk of males for arteriosclerosis and in particularmyocardial infarction (see, Reymer et al. (1995) Nature Genetics10:28-34). In addition, determining changes in allelic frequency canallow the identification of previously unknown sequence variations andultimately a gene or pathway involved in the onset and progression ofdisease.

12. Epigenetics

The methods provided herein can be used to study variations in a targetnucleic acid or protein relative to a reference nucleic acid or proteinthat are not based on sequence, e.g., the identity of bases or aminoacids that are the naturally occurring monomeric units of the nucleicacid or protein. For example, methods provided herein can be used torecognize differences in sequence-independent features such asmethylation patterns, the presence of modified bases or amino acids, ordifferences in higher order structure between the target molecule andthe reference molecule, to generate fragments that are cleaved atsequence-independent sites. Epigenetics is the study of the inheritanceof information based on differences in gene expression rather thandifferences in gene sequence. Epigenetic changes refer to mitoticallyand/or meiotically heritable changes in gene function or changes inhigher order nucleic acid structure that cannot be explained by changesin nucleic acid sequence. Examples of features that are subject toepigenetic variation or change include, but are not limited to, DNAmethylation patterns in animals, histone modification and thePolycomb-trithorax group (Pc-G/tx) protein complexes (see, e.g., Bird,A., Genes Dev., 16:6-21 (2002)).

Epigenetic changes usually, although not necessarily, lead to changes ingene expression that are usually, although not necessarily, inheritable.For example, as discussed further below, changes in methylation patternsis an early event in cancer and other disease development andprogression. In many cancers, certain genes are inappropriately switchedoff or switched on due to aberrant methylation. The ability ofmethylation patterns to repress or activate transcription can beinherited. The Pc-G/trx protein complexes, like methylation, can represstranscription in a heritable fashion. The Pc-G/trx multiprotein assemblyis targeted to specific regions of the genome where it effectivelyfreezes the embryonic gene expression status of a gene, whether the geneis active or inactive, and propagates that state stably throughdevelopment. The ability of the Pc-G/trx group of proteins to target andbind to a genome affects only the level of expression of the genescontained in the genome, and not the properties of the gene products.The methods provided herein can be used with specific cleavage reagentsor specific extension reactions that identify variations in a targetsequence relative to a reference sequence that are based onsequence-independent changes, such as epigenetic changes.

13. Methylation Patterns

The methods provided herein can be used to detect sequence variationsthat are epigenetic changes in the target sequence, such as a change inmethylation patterns in the target sequence. Analysis of cellularmethylation is an emerging research discipline. The covalent addition ofmethyl groups to cytosine is primarily present at CpG dinucleotides(microsatellites). Although the function of CpG islands not located inpromoter regions remains to be explored, CpG islands in promoter regionsare of special interest because their methylation status regulates thetranscription and expression of the associated gene. Methylation ofpromoter regions leads to silencing of gene expression. This silencingis permanent and continues through the process of mitosis. Due to itssignificant role in gene expression, DNA methylation has an impact ondevelopmental processes, imprinting and X-chromosome inactivation aswell as tumor genesis, aging, and also suppression of parasitic DNA.Methylation is thought to be involved in the cancerogenesis of manywidespread tumors, such as lung, breast, and colon cancer, and inleukemia. There is also a relation between methylation and proteindysfunctions (long Q-T syndrome) or metabolic diseases (transientneonatal diabetes, type 2 diabetes).

Bisulfite treatment of genomic DNA can be utilized to analyze positionsof methylated cytosine residues within the DNA. Treating nucleic acidswith bisulfite deaminates cytosine residues to uracil residues, whilemethylated cytosine remains unmodified. Thus, by comparing the sequenceof a target nucleic acid that is not treated with bisulfite with thesequence of the nucleic acid that is treated with bisulfite in themethods provided herein, the degree of methylation in a nucleic acid aswell as the positions where cytosine is methylated can be deduced.

Methylation analysis via restriction endonuclease reaction is madepossible by using restriction enzymes which have methylation-specificrecognition sites, such as HpaII and MSPI. The basic principle is thatcertain enzymes are blocked by methylated cytosine in the recognitionsequence. Once this differentiation is accomplished, subsequent analysisof the resulting fragments can be performed using the methods asprovided herein.

These methods can be used together in combined bisulfite restrictionanalysis (COBRA). Treatment with bisulfite causes a loss in BstUIrecognition site in amplified PCR product, which causes a new detectablefragment to appear on analysis compared to untreated sample. Methodsprovided herein can be used in conjunction with specific cleavage ofmethylation sites to provide rapid, reliable information on themethylation patterns in a target nucleic acid sequence.

14. Resequencing

The dramatically growing amount of available genomic sequenceinformation from various organisms increases the need for technologiesallowing large-scale comparative sequence analysis to correlate sequenceinformation to function, phenotype, or identity. The application of suchtechnologies for comparative sequence analysis can be widespread,including SNP discovery and sequence-specific identification ofpathogens. Therefore, resequencing and high-throughput mutationscreening technologies are critical to the identification of mutationsunderlying disease, as well as the genetic variability underlyingdifferential drug response.

Several approaches have been developed in order to satisfy these needs.Current technology for high-throughput DNA sequencing includes DNAsequencers using electrophoresis and laser-induced fluorescencedetection. Electrophoresis-based sequencing methods have inherentlimitations for detecting heterozygotes and are compromised by GCcompressions. Thus a DNA sequencing platform that produces digital datawithout using electrophoresis can overcome these problems.Matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF MS) measures nucleic acid fragments with digitaldata output. Methods provided herein allow for high-throughput, highspeed and high accuracy in the detection of sequence identity andsequence variations relative to a reference sequence. This approachmakes it possible to routinely use MALDI-TOF MS sequencing for accuratemutation detection, such as screening for founder mutations in BRCA1 andBRCA2, which are linked to the development of breast cancer.

15. Disease Outbreak Monitoring

In times of global transportation and travel outbreaks of pathogenicendemics require close monitoring to prevent their worldwide spread andenable control. DNA based typing by high-throughput technologies enablea rapid sample throughput in a comparatively short time, as required inan outbreak situation (e.g. monitoring in the hospital environment,early warning systems). Monitoring is dependent of the microbial markerregion used, but can facilitate monitoring to the genus, species, strainor subtype specific level. Such approaches can be useful in biodefense,in clinical and pharmaceutical monitoring and metagenomics applications(e.g. analysis of gut flora). Such monitoring of treatment progress orfailure is described in U.S. Pat. Nos. 7,255,992, 7,217,510, 7,226,739and 7,108,974 which are incorporated by reference herein.

16. Vaccine Quality Control and Production Clone Quality Control

Methods provided herein can be used to control the identity ofrecombinant production clones (not limited to vaccines), which can bevaccines or e.g. insulin or any other production clone or biological ormedical product.

17. Microbial Monitoring in Pharmacology for Production Control andQuality

Methods provided herein can be used to control the quality ofpharmacological products by, for example, detecting the presence orabsence of certain microorganism target nucleic acids in such products.

Kits

In some embodiments, provided are kits for carrying out methodsdescribed herein. Kits often comprise one or more containers thatcontain one or more components described herein. A kit comprises one ormore components in any number of separate containers, packets, tubes,vials, multiwell plates and the like, or components may be combined invarious combinations in such containers. One or more of the followingcomponents, for example, may be included in a kit: (i) one or morenucleotides (e.g. terminating nucleotides and/or non-terminatingnucleotides); (ii) one or more nucleotides comprising a capture agent;(iii) one or more oligonucleotides (e.g. oligonucleotide primers, one ormore extension oligonucleotides, oligonucleotides comprising a tag,oligonucleotides comprising a capture agent); (iv) free capture agent(e.g. free biotin); (v) a solid phase (e.g. a bead) comprising a memberof a binding pair (viii); (ix) one or more enzymes (e.g. a polymerase,endonuclease, restriction enzyme, etc.); (x) controls components (e.g.control genomic DNA, primers, synthetic templates, target nucleic acids,etc.) (xi) one or more buffers and (xii) printed matter (e.g.directions, labels, etc).

A kit sometimes is utilized in conjunction with a process, and caninclude instructions for performing one or more processes and/or adescription of one or more compositions. A kit may be utilized to carryout a process (e.g., using a solid phase) described herein. Instructionsand/or descriptions may be in tangible form (e.g., paper and the like)or electronic form (e.g., computer readable file on a tangle medium(e.g., compact disc) and the like) and may be included in a kit insert.A kit also may include a written description of an internet locationthat provides such instructions or descriptions.

EXAMPLES

The examples set forth below illustrate, and do not limit, thetechnology.

Example 1: Pre-PCR Reaction

The presented process provides an alternative biochemistry to theregular PCR, which usually has two gene specific primers amplifying thesame target. The process is suited for the amplification of targetregions e.g. containing a SNP.

Approach 1: This method uses only one primer to extend, see FIG. 1 . Thegene specific extend primer has a 5′ universal PCRTag1R. It is extendedon the genomic DNA. The DNA or the PCR Tag1R gene specific extend primermay be biotinylated, to facilitate clean up of the reaction. Theextended strand is then ligated to a universal phosphorylatedoligonucleotide, which has sequence which is reverse complement of Tag2F(universal PCR primer). To facilitate clean up in the next step, thephosphorylated oligonucleotide has exonuclease resistant nucleotides atits 3′ end. During the exonuclease treatment, all non-ligated extendstrands are digested, whereas ligated products are protected and remainin the reaction. A universal PCR is then performed using Tag1R and theTag2F primers, to amplify multiple targets. An overview of concept-1 isoutlined in FIG. 1 .

Approach 2: In this method, primer extension and ligation takes place inthe same reaction. FIG. 2 shows the use of a biotinylated PCRTag3R genespecific primer as an extension primer. The phosphorylatedoligonucleotide has a gene specific sequence and binds around 40 basesaway from the primer extension site, to the same strand of DNA. Thus,Stoffel DNA polymerase extends the strand, until it reaches thephosphorylated oligonucleotide. Amp ligase (Epicentre) ligates the genespecific sequence of the phosphorylated oligonucleotide to the extendedstrand. The 3′ end of Phospho oligonucleotide has PCRTag4(RC)F as itsuniversal tag. The biotinylated extended strands are then bound tostreptavidin beads. This facilitates clean up of the reaction. GenomicDNA and the gene specific phosphorylated oligonucleotides will getwashed away. A universal PCR is then performed using Tag3R and Tag4F asprimers, to amplify different genes of interest. An overview ofconcept-2 is as shown in FIG. 2 .

The universal PCR products from both the Approach 1 and 2 can beidentified using the post-PCR reaction, as shown in FIG. 3 . SAP wasused to clean up the PCR reaction. Post-PCR reactions were performedusing gene specific oligonucleotides binding just before the SNP and thesingle base extended products were spotted on a chip array and analyzedon mass spectrometry. Alternatively the methods provided herein can beused for post-PCR read-out.

Example 2: Pre-PCR Reaction Materials from Example 1

Approach 1:

1a) Extension: A 90 ul reaction was performed with 18 ng plasmid insert,1× Qiagen PCR buffer with Mg, 2.82 mM of total MgCl2, 10 mM Tris, pH9.5, 50 uM dNTPs, 0.5 uM 5′ PCR tag1R gene specific extension primer,5.76 U Thermosequenase. The thermo cycling conditions used were 2minutes at 94° C. followed by 45 cycles of 10 second denaturation at 94°C.; 10 seconds annealing at 56° C.; 20 seconds extension at 72° C.

1b) Ligation: 5 ul of extended product was ligated with 500 pmols of aphospho oligonucleotide (reverse complement of the Tag2F primer) whichis exonuclease resistant at its 3′end. The extension product andphospho-oligonucleotide were denatured at 65° C./10 minutes, cooledbefore volume made to 50 ul with 50 mM Tris-HCl, pH 7.8, 10 mM MgCl₂, 10mM DTT, 1 mM ATP and 50 U T4 RNA Ligase1. Incubation was carried out at37° C./4 hours, 65° C./20 minutes.

1c) Exonuclease treatment: 10 ul of the ligated product was denatured at95° C./5 minutes, cooled and diluted with 0.5× exonuclease III buffercontaining 20 U exonuclease I and 100 U exonuclease III in a totalvolume of 20 ul. The reaction was incubated at 37° C./4 hours, 80° C./20minutes.

1d) Universal PCR: 2 ul of the exonuclease treated product was amplifiedwith 0.4 uM each of M13 forward and reverse primers in a 25 ul reactioncontaining 1× Qiagen buffer containing 1.5 mM MgCl₂, 200 uM dNTP and0.625 U Hot star DNA polymerase. The thermo cycling conditions used were15 minutes at 94° C., followed by 45 cycles of 30 second denaturation at94° C.; 30 seconds annealing at 55° C. and one minute extension at 72°C.

The primers and PCR tag sequences used were:

Universal Tag 1R (rs10063237)=5′GGAAACAGCTATGACCATG—(GTAATTGTACTGTGAGTGGC) gene specific sequence 3′(SEQ ID NO: 1), Universal Tag2 (RC) F=5′P-CATGICGTITTACAACGTCG*T*G*ddC3′ (SEQ ID NO: 2) (The * represents exonuclease resistant linkagesbetween the nucleotides) Tag1R (M13R)=5′ GGAAACAGCTATGACCATG 3′ (SEQ IDNO: 3) Tag2F (M13F)=5′ CACGACGTTGTAAAACGAC 3′ (SEQ ID NO: 4)rs10063237_E1 (for post-PCR reaction): 5′TCAAAGAATTATATGGCTAAGG 3′ (SEQID NO: 5)

Results from Approach 1 can be seen in FIG. 4 .

Approach 2:

2a) Extension and Ligation: The 20 ul reaction was carried out with16-35 ng genomic DNA, 1× Amp ligase buffer (Epicentre), 200 uM dNTP, 10nM biotinylated extension primer, 50 nM gene specific phosphooligonucleotide, 1 U Stoffel fragment DNA polymerase and 4 U Amp ligase(Epicentre). The thermo cycling conditions used: 5 minutes at 94° C.followed by 19 cycles of 30 second denaturation at 94° C.; 150 secondsannealing at 58.5° C., with a decrease in temperature by at every cycle;45 seconds extension at 72° C. The extension and ligation reaction wastreated with 40 ug of proteinase K at 60° C. for 20 minutes.

2b) Bead Clean up: 15 ul of Dyna beads M-280 streptavidin beads werewashed three times with 1× binding buffer (5 mM Tris-HCl pH 7.5, 1 MNaCl, 0.5 mM EDTA). During all washes, the beads were bound to themagnet and the supernatant then discarded. Two extension reactions werepooled and diluted to get a 1× binding buffer concentration and thenmixed with the beads. The beads were incubated at room temperature for20 minutes, with gentle agitation. The beads were then washed 3 timeswith 1× wash buffer (10 mM Tris, pH 81 mM EDTA) and 2 times with water.The beads were then treated with 0.1N NaOH at room temperature for 10minutes. The beads were then washed 2 times with 1× wash buffer and 2times with water. The beads were finally suspended in 15 ul water.

2c) Universal PCR: 2 ul beads were added to a 25 ul PCR reactioncontaining 1×PCR Gold buffer (Applied Biosystems), 250 uM dNTP, 2.5 mMMgCl₂, and 0.4 uM each of Tag4F and Tag3R primers, 1.25 U AmpliTaq GoldDNA polymerase and 0.05% Tween 20. The thermo cycling conditions usedwere 12 minutes at 94° C. followed by 60 cycles of 30 seconddenaturation at 94° C.; 30 seconds annealing at 68° C.; 45 secondsextension at 72° C., with a final extension of 72° C. for 2 minutes.

The primers and Tag sequences used were:

Universal Tag 3R = (SEQ ID NO: 6)5′ GAGCTGCTGCACCATATTCCTGAAC-gene specific sequence 3′,Universal Tag4 (RC) F =  (SEQ ID NO: 7)5′P-gene specific sequence-GCTCTGAAGGCGGTGTATGACATG G 3′ Tag3R =(SEQ ID NO: 8) 5′ GAGCTGCTGCACCATATTCCTGAAC 3′ Tag4F =  (SEQ ID NO: 9)5′ CCATGTCATACACCGCCTTCAGAGC 3′

Approach 2 gene specific extend primers, phospho oligonucleotides andpost-PCR reaction extension primers are listed in Tables 1, 2 and 3respectively. For Table 1, the PCR tag region is underlined. In Approach2, 5′-Biotinylated and PCR-tagged gene specific-primer is extended ongenomic DNA by Stoffel DNA polymerase and simultaneously ligated to adownstream gene specific PCR-tagged phospho oligonucleotide bound on thesame strand, by Amp Ligase (Epicentre). Results from Approach 2 areshown in FIGS. 5A-5 .

TABLE 1Extension primers used to extend genomic DNA in the extension ligationreaction (non-hybridizing regions are underlined) SEQ ID Primer Name5′Biotin-primer seq NO: 5′BiotinUF rs10005865′Biotin-GAGCTGCTGCACCATATTCCTGAACTCTCAAACTCCAGAGTGGCC 105′BiotinUF rs100120045′Biotin-GAGCTGCTGCACCATATTCCTGAACAGCAGTGCTTCACACACTTTAG 115′BiotinUF rs100140765′Biotin-GAGCTGCTGCACCATATTCCTGAACGTCCTGATTTCTCCTCCAGAG 125′BiotinUF rs100276735′Biotin-GAGCTGCTGCACCATATTCCTGAACCCCTCTTGCATAAAATGTTGCAG 135′BiotinUF rs100287165′Biotin-GAGCTGCTGCACCATATTCCTGAACCATGAAGAGAAATAGTTCTGAGGTTTCC 145′BiotinNewUF rs100632375′Biotin-GAGCTGCTGCACCATATTCCTGAACCTGATAGTAATTGTACTGTGAGTGGC 155′BiotinUF rs10077165′Biotin-GAGCTGCTGCACCATATTCCTGAACCTAAAAACTTATAATTTTAATAGAGGGT 16GCATTGAAG 5′BiotinUF rs101318945′Biotin-GAGCTGCTGCACCATATTCCTGAACACGTAAGCACACATCCCCAG 175′BiotinUF rs10143375′Biotin-GAGCTGCTGCACCATATTCCTGAACGATTTCTATCCTCAAAAAGCTTATGGG 185′BiotinUF rs10157315′Biotin-GAGCTGCTGCACCATATTCCTGAACGATGAATCATCTTACTCTTTAGTATGGTTGC 195′BiotinUF rs101644845′Biotin-GAGCTGCTGCACCATATTCCTGAACCCTGCCCTTTAGACAGGAATC 205′BiotinUF rs102517655′Biotin-GAGCTGCTGCACCATATTCCTGAACCATCTGCCTTGATCTCCCTTC 215′BiotinUF rs102658575′Biotin-GAGCTGCTGCACCATATTCCTGAACCCTTCATGCTCTTCTTCCTGC 225′BiotinUF rs10324265′Biotin-GAGCTGCTGCACCATATTCCTGAACGCTATTTTTATAATATTTATTATTTT 23AAATAATTCAAAATACAAAAGTAACAC 5′BiotinUF rs104955565′Biotin-GAGCTGCTGCACCATATTCCTGAACCTAGACATTGGGAATACATAGGAGTG 245′BiotinUF rs104992265′Biotin-GAGCTGCTGCACCATATTCCTGAACAACTTGTACCCAGATGCAGTC 255′BiotinUF rs105050075′Biotin-GAGCTGCTGCACCATATTCCTGAACCTTCTAAGGCTTCAGGGATGAC 265′BiotinUF rs10630875′Biotin-GAGCTGCTGCACCATATTCCTGAACGTACTTGAAAAGAAGCCCGG 275′BiotinUF rs107323465′Biotin-GAGCTGCTGCACCATATTCCTGAACGATCTCTCTACCACCATCAGGG 285′BiotinNewUF rs107429935′Biotin-GAGCTGCTGCACCATATTCCTGAACAGGAGTCACTACATTCAGGGATG 295′BiotinUF rs108827635′Biotin-GAGCTGCTGCACCATATTCCTGAACGTGTCTCAGGTGAAAGTGACTC 305′BiotinNewUF rs109119465′Biotin-GAGCTGCTGCACCATATTCCTGAACCTTCAGGATTATACTGGCAGTTGC 315′BioinUF rs110332605′Biotin-GAGCTGCTGCACCATATTCCTGAACGCTTTGAATGGTATCACCCTCAC 325′BiotinUF rs112405745′Biotin-GAGCTGCTGCACCATATTCCTGAACAAACGCAGTCATCACTCTCC 335′BiotinUF rs115993885′Biotin-GAGCTGCTGCACCATATTCCTGAACGGGAGCGGGAATCTTAAATCC 345′BiotinUF rs116344055′Biotin-GAGCTGCTGCACCATATTCCTGAACGCAACAGGATTCGACTAAGGC 355′BiotinUF rs12229585′Biotin-GAGCTGCTGCACCATATTCCTGAACCATGTATATAGTTTGGCTAGCAGTGAAAG 365′BiotinUF rs123347565′Biotin-GAGCTGCTGCACCATATTCCTGAACGAATCCTACTCCTAAGGTGATGTTG 375′BiotinUF rs12668865′Biotin-GAGCTGCTGCACCATATTCCTGAACCTTCATCAGCAAGCAACTACATTG 385′BiotinNewUF rs128255665′Biotin-GAGCTGCTGCACCATATTCCTGAACGGGTCCAAAACTGCTCATGTC 395′BiotinUF130233805′Biotin-GAGCTGCTGCACCATATTCCTGAACTTTTTCCATGGCTTTTGGGC 405′BiotinUF rs13932575′Biotin-GAGCTGCTGCACCATATTCCTGAACTGTACAGGCAGGTCTTAGAGATG 415′BiotinUF rs14001305′Biotin-GAGCTGCTGCACCATATTCCTGAACGTAGCCAATTCCTTCAGTGCAG 425′BiotinNewUF rs14904925′Biotin-GAGCTGCTGCACCATATTCCTGAACAGGGCTTGTTTCAGCTTGAG 435′BiotinUF rs15676035′Biotin-GAGCTGCTGCACCATATTCCTGAACCAAAAGTTTTGTTTAGGTGCCTTCC 44

TABLE 2Gene-specific phospho oligonucleotides used to ligate the extended strandin the extension ligation reaction (non-hybridizing regions are underlined)SEQ ID Primer name 5′P-Primer Sequence NO: 5′P rs1000586GGGGAGTGTAGGTTCTGGTACCCAGGCTCTGAAGGCGGTGTATGACATGG 45 5′P rs10012004CATCACCTATATCATTATTTACTAAATTATTTTTTCTTCAAACTGACTTAGGCTCTGAAGGCGGTGTATGACATGG 46 5′P rs10014076CCCTTTTTTCCTAAAAGCCCCCAAACTTTTGGCTCTGAAGGCGGTGTATGACATGG 475′P rs10027673 CTTTTGTGAGCTGGCTTTTGCTCATCTCGCTCTGAAGGCGGTGTATGACATGG 485′P rs10028716CCTATTTGAGTTTTGCTTTTTTGTTTTGGTCTCGGCTCTGAAGGCGGTGTATGACATGG 495′P rs100632371ongGATTTAGACAGAGTCTTACTCTGTCACCAGGGCTCTGAAGGCGGTGTATGACATGG 505′P rs1007716 CTATACTCTTGCTCGTGGAGTTAATCTCAGAGGGCTCTGAAGGCGGTGTATGACATGG51 5′P rs10131894 CTCAGAAGTGTGGAACAGCTGCCCGCTCTGAAGGCGGTGTATGACATGG 525′P rs1014337 CTTGGGACTTCAGGTAGACTTAGTTTGAACATCGCTCTGAAGGCGGTGTATGACATGG53 5′P rs1015731 CCATCTACATTAGCTTACCAGGGCTGCGCTCTGAAGGCGGTGTATGACATGG 545′P rs10164484 CTCTCTAATGTTCCAGAGAAACCCCAGGGCTCTGAAGGCGGTGTATGACATGG 555′P rs10251765 CGTTTTCTTATGTGTCTGGCCTCATCCGCTCTGAAGGCGGTGTATGACATGG 565′P rs10265857 GGAGCGCTCCATGAAACACAACAGGCTCTGAAGGCGGTGTATGACATGG 575′P rs1032426 GTTGACAGTTGATTTTGTAATGCCTCCACGCTCTGAAGGCGGTGTATGACATGG 585′P rs10495556 CGATGTGATCCTGTGTCAAATAATGACGGGCTCTGAAGGCGGTGTATGACATGG 595′P rs10499226CTGAAGGGAATGGCTGGTTTTTAATTTGTAGTGGCTCTGAAGGCGGTGTATGACATGG 605′P rs10505007 GAAGGTGGGATTACGCCTAACTTTAGGGCTCTGAAGGCGGTGTATGACATGG 615′P rs1063087 GACTTCATGGCTGGCAGAAAGCTCTGAAGGCGGTGTATGACATGG 625′P rs10732346 CTGCATTTCTACTGGTAACATGCGCCGCTCTGAAGGCGGTGTATGACATGG 635′PNew rs10742993CTATTCAGGTGTCACTTTTATTATGATTATCTAAGGTCAGTGGCTCTGAAGGCGGTGTA 64 TGACATGG5′P rs10882763 CAGGTCCAGTTCTTGAGTTTCATCCTTTCGCTCTGAAGGCGGTGTATGACATGG 655′P rs109119461ongCCTCTCTGTTTTGTTGAGAAATCCACTCTTGGTCGCTCTGAAGGCGGTGTATGACATGG 665′P rs11033260 GCAAAATGGGTATGGTTTAGCCAGAAACATGGCTCTGAAGGCGGTGTATGACATGG67 5′P rs11240574 GGTGATGGACCCACTGCCTGGCTCTGAAGGCGGTGTATGACATGG 685′P rs11599388 GTGACCTGACACTGGTGGGATGGCTCTGAAGGCGGTGTATGACATGG 695′P rs11634405 GCTTTGTGTGCAAATCACCTATTTTCCTGGCTCTGAAGGCGGTGTATGACATGG 705′P rs1222958 GGTGAGAGAATATGAAAGCAAAACAGCAACCGCTCTGAAGGCGGTGTATGACATGG71 5′P rs12334756 GGGCTATGTAGACACTTCAAAGGTGTTCGCTCTGAAGGCGGTGTATGACATGG72 5′P rs1266886 GTTTGCTCTAGCTCAATGGCCTCTTAAGGCTCTGAAGGCGGTGTATGACATGG73 5′PNew rs12825566CCAACACAGTCATCTGATCCCATCTCCGCTCTGAAGGCGGTGTATGACATGG 74 5′P rs13023380GTAGGCAAGGCTGTTCTTTTTTGTGTTGGCTCTGAAGGCGGTGTATGACATGG 75 5′P rs1393257CCATATGCAGTTTTTGTTTTCCCAGTGCGCTCTGAAGGCGGTGTATGACATGG 76 5′P rs1400130CACCATAATAGTTTATCTGCTTCTACTAAAATTATTATTGGCGCTCTGAAGGCGGTGTA 77 TGACATGG5′PNew rs1490492CCTCAGAATGAAATCATGCTTTTCTGCTAATTTGTAGGCTCTGAAGGCGGTGTATGACA 78 TGG5′P rs1567603 CCTTCAGACATACCTTGGGAAAATGTCAGGCTCTGAAGGCGGTGTATGACATGG 79

TABLE 3Standard post-PCR primers used in the post-PCR assay for the universal PCR readoutSEQ UEP_ UEP_ ID EXT1_ EXT1_ EXT2_ EXT2_ TERM SNP_ID DIR MASSUEP_SEQ 5′-3′ NO: CALL MASS CALL MASS L1 goldPLEX rs10882763 F 4374.9CCTTCTTCATCCCCC 80 G 4662.1 T 4701.9 L2 goldPLEX rs12334756 R 4515GCCCATAAGCCAACA 81 G 4762.2 A 4842.1 L3 goldPLEX rs1014337 F 4627GTCCCAAGGGAGAGC 82 G 4914.2 T 4954.1 L4 goldPLEX rs1063087 R 4875.2GGTAAAGCCCCTCGAA 83 C 5162.4 A 5202.3 L5 goldPLEX rs1000586 R 5027.3CTCCCCACCTGACCCTG 84 G 5274.5 A 5354.4 L6 goldPLEX rs1400130 R 5118.3TTATGGTGTCTTTCCCC 85 T 5389.5 C 5405.5 L7 goldPLEX rs11634405 R 5237.4CAAAGCAGGTGCACGAA 86 G 5484.6 A 5564.5 L8 goldPLEX rs12825566 R 5311.5ACTTCCTCCCTTCTTACT 87 C 5598.7 A 5638.6 L9 goldPLEX rs10251765 F 5448.5CCCTTTTGGCTTCCTGGG 88 G 5735.7 T 5775.6 L10 goldPLEX rs11033260 F 5704.7CCCATTTTGCGCCATTTAT 89 A 5975.9 G 5991.9 L11 goldPLEX rs10495556 F5827.8 GGATCACATCGTGTTAGAC 90 C 6075 T 6154.9 L12 goldPLEX rs10027673 R5867.8 ggAAGACGCTTATCATGGT 91 G 6115 A 6194.9 M1 goldPLEX rs10131894 F6037.9 ccetTGCATGCATGCGCACA 92 C 6285.1 G 6325.1 M2 goldPLEX rs1393257 F6239.1 agGCAATAGAGGGAGTATCA 93 C 6486.3 T 6566.2 M3 goldPLEX rs10164484F 6246.1 aaactTCTCCCTCAGCCTACC 94 A 6517.3 G 6533.3 M4 goldPLEXrs10499226 R 6373.2 CAGAAATACATTTGCCACTAT 95 G 6620.4 C 6660.4 M5goldPLEX rs1007716 R 6446.2 gcGCTGTATCCTCAGAGAGTA 96 G 6693.4 A 6773.3M6 goldPLEX rs10732346 R 6731.4 GGGAGAATGCATTTCTTTTTC 97 T 7002.6 C7018.6 C M7 goldPLEX rs10014076 R 6831.5 GGATACTTCAAGAATAGTAGA 98 G7078.7 A 7158.6 G M8 goldPLEX rs1266886 R 6840.4 cccacTCTATTCCCACGTCAG99 T 7111.7 C 7127.7 CC M9 goldPLEX rs11240574 F 6954.5tttaTTTTTCCATCACACGTA 100 C 7201.7 T 7281.6 TG M10 goldPLEX rs11599388 R7233.7 tttcTAAATCCCCACCCGGCG 101 G 7480.9 A 7560.8 CAG M11 goldPLEXrs1222958 F 7240.7 gCTCTCACCATTAACTATACA 102 A 7511.9 G 7527.9 GCA M12goldPLEX rs10742993 R 7327.8 gttgACAGTTCTCCAAGTCCA 103 T 7599 C 7615 GATH1 goldPLEX rs10505007 F 7398.8 ggattACAGATGCCTTCTTGG 104 A 7670 G 7686GTA H2 goldPLEX rs10063237 R 7722.1 CAATCAAAGAATTATATGGCT 105 G 7969.2 A8049.2 AAGG H3 goldPLEX rs10012004 F 7902.1 ccdtTAACACCTATATGGGTT 106 C8149.3 T 8229.2 TTTG H4 goldPLEX rs13023380 F 7909.2gcagcACAGCCTTGCCTACAA 107 A 8180.4 G 8196.4 TGACA H5 goldPLEX rs1490492F 8098.3 gggCATTCTGAGGAAAATAAT 108 C 8345.5 T 8425.4 GTATG H6 goldPLEXrs10265857 R 8106.3 ggacGAGAGGTCTGAGAGTTT 109 T 8377.5 C 8393.5 CTGAT H7goldPLEX rs1567603 F 8265.4 acATAACTCTCAGATAATTAA 110 C 8512.6 T 8592.5AGTTGT H8 goldPLEX rs1015731 R 8310.5 atgtTAACAGAAAGCACAATA 111 G 8557.7A 8637.6 AAAACA H9 goldPLEX rs10911946 F 8470.5 gggagGAGAGGAACCATAAGA112 C 8717.7 T 8797.6 TATTAG H10 goldPLEX rs10028716 R 8477.5cctggTTTTGTCTTCCCTATT 113 T 8748.7 C 8764.7 TACTGAT H11 goldPLEXrs1032426 F 8672.7 ggacAAAAGTTCTGAATTATT 114 A 8943.9 G 8959.9 TGGTTTG

Example 3: Post-PCR Reaction after Examples 1 and 2

SAP/Post-PCR Reaction: 5 ul Univ PCR was dispensed in a 384 well plateand 2 ul SAP reaction containing 0.6 U SAP (shrimp alkaline phosphatase)were added with incubation at 37° C. for 40 minutes and finallyinactivation of the enzyme at 85° C. for 5 minutes. Extension reagentswere added in 2 ul amounts containing 0.9 mM acyclic terminators and1.353 U post-PCR enzyme. The extension oligonucleotide mixture differedin concentration according to its mass: 0.5 uM of low mass: 4000-5870daltons, 1.0 uM of medium mass: 6000-7350 daltons and 1.5 uM of highmass: 7400-8700 daltons were added in a final volume of 9 ul. Thecycling conditions used for post-PCR reaction were 94° C./30 sec and 40cycles of an 11 temperature cycle (94° C./5 secs and 5 internal cyclesof (52° C./5 sec and 80° C./5 sec) and final extension at 72° C./3minutes.

MALDI-TOF MS: The extension reaction was diluted with 16 ul water and 6mg CLEAN Resin (SEQUENOM) was added to desalt the reaction. It wasrotated for 2 hours at room temperature. 15 nl of the post-PCR reactionwere dispensed robotically onto silicon chips preloaded with matrix(SpectroCHIP®, SEQUENOM). Mass spectra were acquired using a Mass ARRAYCompact Analyzer (MALDI-TOF mass spectrometer, SEQUENOM).

Example 4: Post-PCR Reaction to Increase Multiplexing and Flexibility inSNP Genotyping

The presented process provides a concept for an alternative goldPLEXprimer extension post-PCR format to increase multiplexing andflexibility of SNP genotyping. It utilizes allele specific extensionprimers, with two extension primers per SNP designed to hybridize on theSNP site. Each primer contains a gene and allele specific 3′ nucleotidefor specific hybridization to the SNP site of interest and a varieddefined 5′ nucleotide sequence which corresponds to a mass tag. Thespecificity of the assay is determined by the match of the 3′ end of theprimer to the template, which will only be extended by DNA polymerase ifcorresponding to the specific SNP. An overview of the process isoutlined in FIG. 6 .

The extension primers are extended by dNTP incorporation and terminatedby a ddNTP or alternatively terminated by ddNTP incorporation withoutdNTP extension. One or more dNTP and/or ddNTP used during the extensionreaction are labeled with a moiety allowing immobilization to a solidsupport, such as biotin.

The extension product is subsequently immobilized on a solid support,such as streptavidin coated beads, where only extended/terminatedproducts will bind. Unextended primers and unwanted reaction componentsdo not bind and are washed away.

The 5′ nucleotide sequence or an alternative group which corresponds toa mass tag is cleaved from the extension product, leaving the 3′ sectionof the extension product bound to the solid support. The cleavage can beachieved with a variety of methods including enzymatic, chemical andphysical treatments. The possibility outlined in this example utilizesEndonuclease V to cleave a deoxyinosine within the primer. The reactioncleaves the second phosphodiester bonds 3′ to deoxyinosine releasing anoligonucleotide mass tag.

The 5′ nucleotide sequence (mass tag) is then transferred to a chiparray and analyzed by mass spectrometry (e.g. MALDI-TOF MS). Thepresence of a mass signal matching the tag's mass indicates an allelespecific primer was extended and therefore the presence of that specificallele.

Example 5: Endonuclease V Cleavage of Deoxyinosine

Prior to the extension reaction a 35 plex PCR was carried out in a 5 μlreaction volume using the following reagents; 5 ng DNA, 1×PCR buffer,500 μM each dNTP, 100 nM each PCR primer (as listed in Table 4), 3 mMMgCl₂, and 0.15 U Taq (SEQUENOM). Thermocycling was carried out usingthe following conditions: 7 minutes at 95° C.; followed by 45 cycles of20 seconds at 95° C., 30 seconds at 56° C. and 1 minute at 72° C.; andconcludes with 3 minutes at 72° C.

The PCR reaction was treated with SAP (shrimp alkaline phosphatase) todephosphorylate unincorporated dNTPs. A 2 μl mixture containing 0.6 USAP was added to the PCR product and then subjected to 40 minutes at 37°C. and 5 minutes at 85° C.

Extension reaction reagents were combined in a 3 μl volume, which wasadded to the SAP treated PCR product. The total extension reactioncontained the following reagents; 1× goldPLEX buffer, 17 μM each biotinddNTP, 0.8 μM each extension primer (listed in Table 5) and 1×post-goldPLEX enzyme.

Thermocycling was carried out using a 200 cycle program consisting of 2minutes at 94° C.; followed by 40 cycles of 5 seconds at 94° C.,followed by 5 cycles of 5 seconds at 52° C., and 5 seconds at 72° C.;and concludes with 3 minutes at 72° C. Extension primer sequencescontaining the mass tags and resulting masses of the cleaved productscorresponding to specific alleles are listed in Table 5.

Solulink magnetic streptavidin beads were conditioned by washing threetimes with 50 mM Tris-HCl pH 7.5, 1M NaCl, 0.5 mM EDTA, pH 7.5. Theextension reaction was then combined with 300 μg conditioned beads.Beads were incubated at room temperature for 30 minutes with gentleagitation and then pelleted using a magnetic rack. The supernatant wasremoved. Subsequently the beads were washed 3 times with 50 mM Tris-HCl,1M NaCl, 0.5 mM EDTA, pH 7.5 and 3 times with water. For each wash stepthe beads were pelleted and the supernatant removed. The mass tags werecleaved from the extension product by addition of a solution containing30 U Endonuclease V and 0.4× buffer 4(NEB) and incubation at 37° C. for1 hour. After incubation the magnetic beads were pelleted using amagnetic rack and the supernatant containing the mass tag products wasremoved.

Desalting was achieved by the addition of 6 mg CLEAN Resin (SEQUENOM).15 nl of the cleavage reactions were dispensed robotically onto siliconchips preloaded with matrix (SpectroCHIP®, SEQUENOM). Mass spectra wereacquired using a MassARRAY Compact Analyser (MALDI-TOF mass spectrometer(SEQUENOM). FIG. 7 shows MALDI-TOF MS spectra for 35 plex genotypingusing the post-PCR readout as presented herein.

TABLE 4 PCR primers used in this study SEQ ID SEQ ID SNP IDForward Primer NO: Reverse Primer NO: rs11155591ACGTTGGATGAAAGGCTGATCCAGGTCATC 115 ACGTTGGATGTTCTCTTCAAACCTCCCATC 150rs12554258 ACGTTGGATGTTGAGACACGGCACAGCGG 116ACGTTGGATGTTTTCCTCTTCCTACCCCTC 151 rs12162441ACGTTGGATGAAGGTAGGCCTTTAGGAGAG 117 ACGTTGGATGTGGCAACACACGACTGTACT 152rs11658800 ACGTTGGATGATGCACAATCGTCCTACTCC 118ACGTTGGATGTGCTTCCCAGGTCACTATTG 153 rs13194159ACGTTGGATGTGAGCCAGGGATATCCTAAC 119 ACGTTGGATGTCCATGAGTGCAGGACTACG 154rs1007716 ACGTTGGATGTAATAGAGGGTGCATTGAAG 120ACGTTGGATGCTCCACGAGCAAGAGTATAG 155 rs11637827ACGTTGGATGAAAGAGAGAGAGATCCCTG 121 ACGTTGGATGATCCCATACGGCCAAGAAGA 156rs13188128 ACGTTGGATGCACTAATAAAGGCAGCCTGT 122ACGTTGGATGATGAGTAACGCTTGGTGCTG 157 rs1545444ACGTTGGATGGGCTCTGATCCCTTTTTTTAG 123 ACGTTGGATGTGGTAGCCTCAAGAATGCTC 158rs1544928 ACGTTGGATGGCTTTTCCTCTTCTTTGGTAG 124ACGTTGGATGGAATGTGTAAAACAAACCAG 159 rs11190684ACGTTGGATGTCTCAGTTCCAACTCATGCC 125 ACGTTGGATGTGAGCCATGTAGAGACTCAG 160rs12147286 ACGTTGGATGAGAATGTGCCAAAGAGCAG 126ACGTTGGATGTCTGCATCCCTTAGGTTCAC 161 rs11256200ACGTTGGATGCCTTATTGGATTCTATGTCCC 127 ACGTTGGATGACCAAGCACTGTACTTTTC 162rs1124181 ACGTTGGATGACTTGGCGAGTCCCCATTTC 128ACGTTGGATGTTAATATAGTCCCCAGCCAC 163 rs1392592ACGTTGGATGTCTTGTCTCTTACCTCTCAG 129 ACGTTGGATGCTGTGCTGACTGAGTAGATG 164rs1507157 ACGTTGGATGTGAGGATTAAAGGATCTGGG 130ACGTTGGATGATCTTTGAAGGCTCCTCTGG 165 rs1569907ACGTTGGATGGAGGCTCCTCTACACAAAAG 131 ACGTTGGATGGCATGTCCCTATGAGATCAG 166rs1339007 ACGTTGGATGTTGCTCTAAGGTGGATGCTG 132ACGTTGGATGTTAGGCACCCCAAGTTTCAG 167 rs1175500ACGTTGGATGGTTTACAACCTGTGGCAGAC 133 ACGTTGGATGTGTAGCATGTCAGCCATCAG 168rs11797485 ACGTTGGATGGAAAGTGACCCATCAAGCAG 134ACGTTGGATGGTAGTTGCTTGTGGTTACCG 169 rs1475270ACGTTGGATGCTATGGGGAACTGAATAAGTG 135 ACGTTGGATGGAGCAATTCATTTGTCTCC 170rs12631412 ACGTTGGATGCAAACTATTGACTGGTCATGG 136ACGTTGGATGTTTTGTTGTTTGGGCATTGG 171 rs1456076ACGTTGGATGGCAGAGGTTTGAGAAAAGAG 137 ACGTTGGATGGTTCCCATCCAGTAATGGAG 172rs12958106 ACGTTGGATGGTATATGCCTGTATGTGGTC 138ACGTTGGATGCCAACAGTTTTTCTTTAAGGG 173 rs1436633ACGTTGGATGGAGGGAAAGACCTGCTTCTA 139 ACGTTGGATGAGAAGCTCCGAGAAAAGGTG 174rs1587543 ACGTTGGATGGAGAAGGCTTTCCAGAATTTG 140ACGTTGGATGTATAGCCATTACTGGGCTTG 175 rs10027673ACGTTGGATGCAAAAGCCAGCTCACAAAAG 141 ACGTTGGATGCCCTCTTGCATAAAATGTTGC 176rs12750459 ACGTTGGATGTTTTGGGCCCCTCCATATTC 142ACGTTGGATGCTCCATGCAAGGCTGTGGC 177 rs13144228ACGTTGGATGTGGATATGCTGAATTTGAGG 143 ACGTTGGATGCGTTATCAAGGACTTTGTGC 178rs11131052 ACGTTGGATGCTTTTGTCCATGTTTGGCAG 144ACGTTGGATGGAGGTTATCTTATTGTAACGC 179 rs1495805ACGTTGGATGAGGACAGTTGTCGTGAGATG 145 ACGTTGGATGAGACTGTCCTTTCCCAGGAT 180rs1664131 ACGTTGGATGCTGAGGCTGGGTAACTTATC 146ACGTTGGATGTCATCAGAAGCAGATGCTGG 181 rs1527448ACGTTGGATGGCCCTTGGCACATAGTACTG 147 ACGTTGGATGCCATACGTTCAAGGATTGGG 182rs11062992 ACGTTGGATGTTGGTTATAGAGCGTCCCTG 148ACGTTGGATGAGGTGTGCAAGTGTCAGAAG 183 rs12518099ACGTTGGATGACCCCTTACTCCAATAAGTC 149 ACGTTGGATGGTATATCATGTCCAGTGAAG 184

TABLE 5 Extension primers and mass tags released after cleavage* SEQ SEQID Mass Tag ID SNP ID Extension Primer Sequence NO: Sequence NO: Massrs11155591_a CCACCGCCTCCICCTCCCATCTCCACCCTCTA 185 CCACCGCCTCCIC 2553802.49 rs11155591_g CCACCGCCTACICCTCCCATCTCCACCCTCTG 186 CCACCGCCTACIC256 3826.52 rs12554258_c CCACAGCCTACICTTCCTACCCCTCCAGCCGC 187CCACAGCCTACIC 257 3850.54 rs12554258_t CCACAGCATACICTTCCTACCCCTCCAGCCGT188 CCACAGCATACIC 258 3874.57 rs12162441_cCAACAGCACAAITTGCTATCCCCACAATTACC 189 CAACAGCACAAIT 259 3922.62rs12162441_t CAACAGAACAAITTGCTATCCCCACAATTACT 190 CAACAGAACAAIT 2603946.64 rs11658800_c CAAAAGAACAAITGAAACTGCAGACTCTTCCC 191 CAAAAGAACAAIT261 3970.67 rs11658800_t CAAAAGAAAAAITGAAACTGCAGACTCTTCCT 192CAAAAGAAAAAIT 262 3994.69 rs13194159_c AATAAGAAGAAICGTCTGATTGGCTTTAGTTC193 AATAAGAAGAAIC 263 4010.69 rs13194159_tGATAAGAAGAAICGTCTGATTGGCTTTAGTTT 194 GATAAGAAGAAIC 264 4026.69rs1007716_c AATAGCGAGAAIGCTGTATCCTCAGAGAGTAC 195 AATAGCGAGAAIG 2654042.69 rs1007716_t AATAGCGAGAGIGCTGTATCCTCAGAGAGTAT 196 AATAGCGAGAGIG266 4058.69 rs11637827_a CCACCCCCGCCCITTCTCCCACAGTAAACTTCCA 197CCACCCCCGCCCIT 267 4091.68 rs11637827_gCCACCACCGCCCITTCTCCCACAGTAAACTTCCG 198 CCACCACCGCCCIT 268 4115.70rs13188128_c CCACCGCACTACICTCTTCTGCTTCATATTTCAC 199 CCACCGCACTACIC 2694139.73 rs13188128_g CCACAGCACTACICTCTTCTGCTTCATATTTCAG 200CCACAGCACTACIC 270 4163.75 rs1545444_aCAACAGCACCACITTCATTATTTCACTCAAGCGA 201 CAACAGCACCACIT 271 4187.78rs1545444_g CAACAGCAACACITTCATTATTTCACTCAAGCGG 202 CAACAGCAACACIT 2724211.80 rs1544928_a CAACAGCTACAAIAAACAAACCAGAAAGTCACTA 203CAACAGCTACAAIA 273 4235.83 rs1544928_gCAACAGATACAAIAAACAAACCAGAAAGTCACTG 204 CAACAGATACAAIA 274 4259.85rs11190684_c CAAAAGATACAAIATGTAGAGACTCAGTCTCTTC 205 CAAAAGATACAAIA 2754283.88 rs11190684_g CAAAAGATAGAAIATGTAGAGACTCAGTCTCTTG 206CAAAAGATAGAAIA 276 4323.90 rs12147286_cCAAAAGAGAGAAITGCAAATTAGATTTGTCAGGC 207 CAAAAGAGAGAAIT 277 4339.90rs12147286_t CAGAAGAGAGAAITGCAAATTAGATTTGTCAGGT 208 CAGAAGAGAGAAIT 2784355.90 rs11256200_a CAGAAGAGAGAGITATGTCTTATTCTTCTTCACCA 209CAGAAGAGAGAGIT 279 4371.90 rs11256200_gCAGGAGAGAGAGITATGTCTTATTCTTCTTCACCG 210 CAGGAGAGAGAGIT 280 4387.90rs1124181_c CCACCCACCGCCCITAGTCCCCAGCCACTATAAAAC 211 CCACCCACCGCCCIT 2814404.89 rs1124181_g CCACCCGCCGCCCITAGTCCCCAGCCACTATAAAAG 212CCACCCGCCGCCCIT 282 4420.89 rs1392592_cCCACCCGCCGCTCITTCCCAAAGTTGAGGGACTTAC 213 CCACCCGCCGCTCIT 283 4435.90rs1392592_t CCACTCGCCGCTCITTCCCAAAGTTGAGGGACTTAT 214 CCACTCGCCGCTCIT 2844450.91 rs1507157_c CCACGCGCCCTACIAAGGCTCCTCTGGGGCACAAGC 215CCACGCGCCCTACIA 285 4468.94 rs1507157_tCAACGCGCACTACIAAGGCTCCTCTGGGGCACAAGT 216 CAACGCGCACTACIA 286 4516.99rs1569907_a CAACAAGCACTACIGGGTTTTGTTGTGCCAGTAGAA 217 CAACAAGCACTACIG 2874541.01 rs1569907_g CAACAAGCAATACIGGGTTTTGTTGTGCCAGTAGAG 218CAACAAGCAATACIG 288 4565.04 rs1339007_cCAAGAAGAAATAAICTGCCAATTAATCATCAACTCTC 219 CAAGAAGAAATAAIC 289 4613.09rs1339007_t AAAGAAGAAATAAICTGCCAATTAATCATCAACTCTT 220 AAAGAAGAAATAAIC290 4637.11 rs1175500_a GAAGAAGACATAAIATGTCAGCCATCAGCCTCTCACA 221GAAGAAGACATAAIA 291 4653.11 rs1175500_gGAAGAAGACATAGIATGTCAGCCATCAGCCTCTCACG 222 GAAGAAGACATAGIA 292 4669.11rs11797485_c GAAGAGGACGTAGIGCTCTTATATCTCATATGAACAC 223 GAAGAGGACGTAGIG293 4717.11 rs11797485_g GAGGAGGACGTAGIGCTCTTATATCTCATATGAACAG 224GAGGAGGACGTAGIG 294 4733.11 rs1475270_cCCACGCTCCTCTACIACTTTTCATGGTTATTCTCAGTC 225 CCACGCTCCTCTACIA 295 4748.12rs1475270_t CCGCGCTCCTCTACIACTTTTCATGGTTATTCTCAGTT 226 CCGCGCTCCTCTACIA296 4764.12 rs12631412_c CCACGCGCACCAACITGTTTTGTTTGTTTTGTTTTTTC 227CCACGCGCACCAACIT 297 4782.15 rs12631412_tCCACGCGCGCCAACITGTTTTGTTTGTTTTGTTTTTTT 228 CCACGCGCGCCAACIT 298 4798.15rs1456076_c CCACGCGAGTCAACICCATCCAGTAATGGAGTACAGTC 229 CCACGCGAGTCAACIC299 4822.17 rs1456076_g CCACGAGAGTCAACICCATCCAGTAATGGAGTACAGTG 230CCACGAGAGTCAACIC 300 4846.20 rs12958106_aCCACGAGAGTCAACIAGTTTTTCTTTAAGGGGAGTAGA 231 CCACGAGAGTCAACIA 301 4870.22rs12958106_g CAACGAGAGTAAACIAGTTTTTCTTTAAGGGGAGTAGG 232 CAACGAGAGTAAACIA302 4918.27 rs1436633_c CAAAGAGAATAAACIGGACAAAGATGAGTGCGTATATC 233CAAAGAGAATAAACIG 303 4942.30 rs1436633_aCAAAGAGAATAAAAIGGACAAAGATGAGTGCGTATATT 234 CAAAGAGAATAAAAIG 304 4966.32rs1587543_a CAAAGAGAATAGAAIGGCTTGGGGTCCCCATTAAAGCGA 235 CAAAGAGAATAGAAIG305 4982.32 rs1587543_g CAGAGAGAATAGAAIGGCTTGGGGTCCCCATTAAAGCGG 236CAGAGAGAATAGAAIG 306 4998.32 rs10027673_cAAGAGCGAGAGAGAITACTAAAGACGCTTATCATGGTC 237 AAGAGCGAGAGAGAIT 307 5014.32rs10027673_t AGGAGCGAGAGAGAITACTAAAGACGCTTATCATGGTT 238 AGGAGCGAGAGAGAIT308 5030.32 rs12750459_c CGGAGAGAGAGGAGITGCAAGGCTGTGGCTGGACAAGAC 239CGGAGAGAGAGGAGIT 309 5046.32 rs12750459_tCGGAGAGGGAGGAGITGCAAGGCTGTGGCTGGACAAGAT 240 CGGAGAGGGAGGAGIT 310 5062.31rs13144228_c CCCGCTCCGCCAGTCIATTCTATATTAGAACAACTCTCTTC 241CCCGCTCCGCCAGTCIA 311 5078.31 rs13144228_tCCACGCGCGCCAGTCIATTCTATATTAGAACAACTCTCTTT 242 CCACGCGCGCCAGTCIA 3125127.35 rs11131052_c CCACGCGCGACAGACITAACGCATATGCACATGCACACATC 243CCACGCGCGACAGACIT 313 5151.38 rs11131052_tCCACGCGAGACAGACITAACGCATATGCACATGCACACATT 244 CCACGCGAGACAGACIT 3145175.40 rs1495805_c CAACGCGAGACAGACITGTCCTTTCCCAGGATGCTCAAAGC 245CAACGCGAGACAGACIT 315 5199.43 rs1495805_tCAACGCGAGACAGAAITGTCCTTTCCCAGGATGCTCAAAGT 246 CAACGCGAGACAGAAIT 3165223.45 rs1664131_g CAACGAGAGACAGTAIAGCAGATGCTGGCCCCATGCTTCAG 247CAACGAGAGACAGTAIA 317 5247.48 rs1664131_tCAACGAGAGAAAGTAIAGCAGATGCTGGCCCCATGCTTCAT 248 CAACGAGAGAAAGTAIA 3185271.50 rs1527448_c CAAGGAGAGAAAGAAITAATAGTACAACAGCTATCAATTAC 249CAAGGAGAGAAAGAAIT 319 5311.53 rs1527448_tCAAGGAGAGAGAGAAITAATAGTACAACAGCTATCAATTAT 250 CAAGGAGAGAGAGAAIT 3205327.53 rs11062992_a CAAGGAGAGAGAGAGITGTGCAAGTGTCAGAAGATGAACAA 251CAAGGAGAGAGAGAGIT 321 5343.53 rs11062992_gCGAGGAGAGAGAGAGITGTGCAAGTGTCAGAAGATGAACAG 252 CGAGGAGAGAGAGAGIT 3225359.53 rs12518099_c CCACCTACCACCAGTCIGAAGAAATAAGAAACATTGAGACAC 253CCACCTACCACCAGTCIG 323 5375.52 rs12518099_tCCACATACCACCAGTCIGAAGAAATAAGAAACATTGAGACAT 254 CCACATACCACCAGTCIG 3245399.55 *SNP specific nucleotides are underlined, mass tags areunderlined and “I” refers to deoxyinosine.

Example 6: RNase A Cleavage of Ribonucleotide

Materials and Methods

Prior to the extension reaction a 2-plex PCR was carried out in a 5 μlreaction volume using the following reagents; 2 ng DNA, 1.25× HotStarTaq buffer, 500 μM each dNTP, 100 nM each PCR primer (as listed in Table1), 3.5 mM MgCl₂, and 0.15 U HotStar Taq (Qiagen). Thermocycling wascarried out using the following conditions: 15 minutes at 95° C.;followed by 45 cycles of 20 seconds at 95° C., 30 seconds at 56° C. and1 minute at 72° C.; and concludes with 3 minutes at 72° C. The PCRreaction was treated with SAP (shrimp alkaline phosphatase) todephosphorylate unincorporated dNTPs. A 2 μl mixture containing 0.3 USAP was added to the PCR product and then subjected to 40 minutes at 37°C. and 5 minutes at 85° C.

TABLE 6 PCR primers used SEQ SEQ Forward ID Reverse ID SNP ID Primer NO:Primer NO: rs1000586 ACGTTGGATGTA 325 ACGTTGGATGTC 327 CCAGAACCTACATCAAACTCCAGA CTCCCC GTGGCC rs10131894 ACGTTGGATGAC 326 ACGTTGGATGAG 328GTAAGCACACAT CTGTTCCACACT CCCCAG TCTGAG

Extension reaction reagents were combined in a 2 μl volume, which wasadded to the SAP treated PCR product. The extension reaction containedthe following reagents; 21 μM each biotin ddNTP, 1 μM each extensionprimer including a ribonucleotide for subsequent RNase A cleavage(listed in Table 7) and 1.25 U Thermo Sequenase. Thermocycling wascarried out using the following cycling conditions: 2 minutes at 94° C.;followed by 100 cycles of 5 seconds at 94° C., 5 seconds at 52° C., and5 seconds at 72° C.; and concludes with 3 minutes at 72° C. Removal ofunbound nucleotides was carried out using the QIAquick NucleotideRemoval Kit (Qiagen) as recommended by the manufacturer.

The eluted extension reaction was then combined with 30 lig preparedDynabeads M-280 Streptavidin beads (Dynal) (washed three times with 5 mMTris-HCl pH 7.5, 1M NaCl, 0.5 mM EDTA). Beads were incubated at roomtemperature for 15 minutes with gentle agitation and then pelleted usinga magnetic rack. The supernatant was removed. Subsequently the beadswere washed 6 times with 5 mM Tris-HCl pH 7.5, 1 M NaCl, 0.5 mM EDTA.For each wash step the beads were pelleted and the supernatant removed.

The mass tags were cleaved from the extension product by addition ofRNase A and incubation at 37° C. for 1 hour. After incubation themagnetic beads were pelleted using a magnetic rack and the supernatantcontaining the mass tag products was removed. Desalting was achieved bythe addition of 6 mg CLEAN Resin (SEQUENOM).

15 nl of the cleavage reactions were dispensed robotically onto siliconchips preloaded with matrix (SpectroCHIP®, SEQUENOM). Mass spectra wereacquired using a MassARRAY Compact Analyser (MALDI-TOF massspectrometer, SEQUENOM).

Extension primer sequences containing the mass tags and resulting massesof the cleaved products corresponding to specific alleles are listed inTable 7. Example spectra are shown in FIG. 8 . For each of the two SNPsboth homozygous as well as a heterozygous sample are displayed and showa clear distinction of the corresponding mass tags.

TABLE 7 Extension primers and mass tags released after cleavage SEQ SEQID Mass Tag ID Assay Name ExtensionPrimer Sequence NO: Sequence NO: Massrs1000586_C TTTCTCCCC ACCTGACCCTGC 329 TTTCTCCCC 333 2697.73 rs1000586_TTTTTCTCCCC ACCTGACCCTGT 330 TTTTCTCCCC 334 3001.93 rs10131894_CTTATTCCCAGGU GCATGCATGCGCACAC 331 TTATTCCCAGGU 335 3694.37 rs10131894_GTTATTTCCCAGGU GCATGCATGCGCACAG 332 TTATTTCCCAGGU 336 3998.57

In Table 7, ribonucleotides are highlighted in bold, SNP specificnucleotides are underlined and mass tags are underlined. In FIG. 8 ,MALDI-TOF MS spectra are shown for genotyping of rs1000586 andrs10131894.

Example 7: Mass Tag Design

Mass Tags were designed to be at least 16 Daltons apart to avoid anyoverlap with potential salt adducts, and so a double charge of any masssignal would not interfere with a mass tag signal. The calculation ofthe mass tags must take into account the deoxyinosine and the nucleotide3′ to the deoxyinosine.

Nucleotide mass tags: MALDI-TOF flight behavior was examined foroligonucleotides which correspond to the mass tags used in a 70 plex(FIGS. 9 and 10 ) and 100 plex assay (FIGS. 11A and B).

All oligonucleotides corresponding to a 70 plex assay were called by thestandard SEQUENOM Typer 3.4 software using the three parameters; area,peak height and signal-to-noise ratio at a comparable level (FIG. 9 ).Using oligonucleotides representing a 70 plex assay, the area value ofeach peak correlates to the sequence composition of thatoligonucleotide. The higher percentage of guanidine and cytosinenucleotides results in larger area values; whereas the percentage ofadenosine corresponds with lower area values (FIG. 10 ). Usingoligonucleotides representing a 100 plex assay we examined the effectsof oligonucleotide concentration (10, 5, 2.5 and 1 pmol finalconcentration per oligonucleotide) on signal-to-noise ratio (FIG. 11B).The lower oligonucleotide concentrations of 2.5 and 1 pmol gaveconsistently higher signal-to-noise ratio values than oligonucleotidesconcentrations of 10 and 5 pmol. This observation was confirmed bymanual observation of the peaks seen in Typer 3.4. However, the fouroligonucleotides concentrations gave comparable area values (data notshown).

Example 8: Extension Primer Design and dNTP/ddNTP Incorporation

Extension primers were designed using SEQUENOM's Assay Design softwareutilizing the following parameters SBE Mass Extend/goldPLEX extension,primer lengths between 20 and 35 bases (and corresponding mass window),and a minimum peak separation of 10 Daltons for analytes (the minimumpossible) and 0 Daltons for mass extend primers.

Extension oligonucleotide and ddNTP role in extension reaction: Toinvestigate the effects of extension oligonucleotide (with/withoutdeoxyinosine nucleotide) and ddNTP composition (with/without biotinmoiety) upon primer extension, we investigated extension rates of a 5plex (FIG. 12 ). Assays generally show the best extension rates usingunmodified extension oligonucleotides and ddNTPs. Extensionoligonucleotides containing a deoxyinosine showed no significantreduction in extension rate. However, when using a ddNTP including abiotin moiety a reduction in extension rate was seen in all assays, whenusing either type of extension oligonucleotide.

Biotinylated dNTP/ddNTP extension: To compare the effects of extendingby a single biotinylated ddNTP or a biotinylated dNTP and terminated byan unmodified ddNTP, we compared extension rates in a 7 plex and 5 plex.The 7 plex was extended by a biotinylated ddCTP or biotinylated dCTP anda ddATP, ddUTP, or ddGTP. The 5 plex was extended by a biotinylatedddUTP or biotinylated dUTP and a ddATP, ddCTP, or ddGTP. The experimentalso compared two concentrations of biotinylated dNTP or ddNTP, either210 or 420 pmol.

In both plexes, and in all individual assays extension rates whenextended by a biotinylated dNTP and terminated by an unmodified ddNTPwere significantly decreased when compared to extending by a singlebiotinylated ddNTPs (FIG. 13 ).

These results indicated that extension with a single biotinylated ddNTPsgives greater extension efficiency.

PCR Amplification

Prior to the extension reaction a PCR was carried out in a 5 μl reactionvolume using the following reagents; 5 ng DNA, 1×PCR buffer, 500 μM eachdNTP, 100 nM each PCR primer, 3 mM MgCl₂, and 0.15 U Taq (SEQUENOM).

Thermocycling was carried out using the following conditions: 7 minutesat 95° C.; followed by 45 cycles of 20 seconds at 95° C., 30 seconds at56° C. and 1 minute at 72° C.; and concludes with 3 minutes at 72° C.

SAP Treatment

The PCR reaction was treated with SAP (shrimp alkaline phosphatase) todephosphorylate unincorporated dNTPs. A 2 μl mixture containing 0.6 USAP was added to the PCR product and then subjected to 40 minutes at 37°C. and 5 minutes at 85° C. in a Thermocycler.

Extension Reaction

Extension reaction reagents were combined in a 3 μl volume, which wasadded to the SAP treated PCR product. The total extension reactioncontained the following reagents; 1× goldPLEX buffer, 0.2 μl of 250 μMstock each biotinylated ddNTP (50 pmol final), 0.8 μl of 2.5 μM solutioneach extension primer (2 pmol final) (IDT), and 0.05 μl goldPLEX enzyme(SEQUENOM).

Thermocycling was carried out using a 300 cycle program consisting of: 2minutes at 94° C.; followed by 60 cycles of; 5 seconds at 94° C.followed by 5 cycles of 5 seconds at 52° C. and 5 seconds at 80° C.; andconcludes with 3 minutes at 72° C.

Capture

For conditioning magnetic streptavidin beads were washed two times with100 μl of 50 mM Tris-HCl, 1M NaCl, 0.5 mM EDTA, pH 7.5. The extensionreaction was combined with 50 μg (5 μl) conditioned beads. Beads wereincubated at room temperature for 1 hour with gentle agitation and thenpelleted using a magnetic rack. The supernatant was removed.Subsequently the beads were washed 3 times with 100 μl of 50 mMTris-HCl, 1 M NaCl, 0.5 mM EDTA, pH 7.5 and 3 times with 100 μl ofwater. For each wash step the beads were pelleted and the supernatantremoved.

MALDI-TOF

Desalting was achieved by the addition of 6 mg CLEAN Resin (SEQUENOM).15 nl of the cleavage reactions was dispensed robotically onto siliconchips preloaded with matrix (SpectroCHIP®, SEQUENOM). Mass spectra wereacquired using a MassARRAY Compact Analyser (MALDI-TOF massspectrometer).

Example 9: Enzyme, Buffer, Oligonucleotide and Biotin ddNTP Titration

Enzyme Titration: The amount of post-PCR enzyme used in the extensionreaction was examined. The standard PCR, extension, andimmobilization/cleavage conditions (as outlined in the protocol inExample 8) were used except for the enzyme. The amount of enzyme usedresulted in no difference in either manual calls or signal-to-noiseratio values for individual assays (FIG. 14 ).

Buffer Titration: The amount of goldPLEX buffer used in the extensionreaction was examined. The standard PCR, extension, andimmobilization/cleavage conditions (as outlined in the protocol inexample 8) were used except for adjusting the amount of buffer. Theamount of buffer used resulted in no difference in either manual callsor signal-to-noise ratio values for individual assays (FIG. 15 ).

Oligonucleotide Titration: The amount of oligonucleotide used in theextension reaction was examined. The standard PCR, extension, andimmobilization/cleavage conditions (as outlined in the protocol section)were used except for adjusting the amount of oligonucleotide.

In the initial experiment (FIG. 16 ) final amounts of 15 pmol, 10 pmoland 5 pmol of each oligonucleotide were tested. The 10 and 15 pmolamounts gave similar results, but 5 pmol gave significantly more manualand software genotype calls. This can be seen by observingsignal-to-noise ratio values (FIG. 9 ), where poorly performing assaysshowing an increased signal-to-noise ratio when using lower amounts ofoligonucleotide.

In follow-up experiments final amounts of 5 pmol, 2.5 pmol and 1 pmol ofeach oligonucleotide were tested (FIG. 17 ). The results for all threeamounts gave similar results as assessed by signal-to-noise ratio andmanual genotype calls. However, three individual assays, for which peakswere clearly seen when concentrations of 2.5 or 1 pmol were used, weredifficult to call due to low intensity when a final concentration of 5pmol was used. When using two 70 plex assays comparing final amounts of2 pmol, 1 pmol and 0.5 pmol of each oligonucleotide the same amount ofmanual calls were seen for all concentrations. However, greatersignal-to-noise ratios were seen when more oligonucleotide was used(FIGS. 18 and 19 ).

These results show the optimal amount of each oligonucleotide to be 2pmol when using a 70 plex assay. However, similar results were seen withfinal amounts of each oligonucleotide ranging from 0.5 to 5 pmol.

Biotinylated ddNTP concentration: The amount of biotinylated ddNTP usedin the extension reaction was examined. The standard PCR, extension, andimmobilization/cleavage conditions (as outlined in the protocol inExample 8) were used except for adjusting the amount of biotinylatedddNTP.

In the initial experiment final amounts of 100, 200, 300 and 400 pmol ofeach biotinylated ddNTP in each extension reaction were tested. Manualcalls and signal-to-noise ratio (FIG. 20 ), show similar results wereseen with all test amounts of biotinylated ddNTP.

To further investigate the amount of biotinylated ddNTP needed in eachextension reaction, an experiment compared 50 and 100 pmol of eachbiotinylated ddNTP in an alternative 70 plex assay.

These assays again show no difference in manual calls or signal-to-noiseratio (FIG. 21 ). This indicates 50 pmol of each biotinylated ddNTP issufficient to get an optimal extension reaction when using a 70 plexassay.

Example 10: Capture and Cleavage Optimization

Immobilization and Oligonucleotide Cleavage: Binding capacity ofmagnetic streptavidin beads. Comparison of Solulink and Dynabeads MyOneC1 magnetic streptavidin beads to capture biotinylated oligonucleotidefollowed the capture protocol as described in Example 8. The experimentuses two oligonucleotides which correspond to extension products for thetwo possible alleles for an assay designed for SNP rs1000586. Theoligonucleotides contain a deoxyinosine nucleotide and 3′ biotinylatednucleotide. The oligonucleotides are bound to the magnetic streptavidinin the presence of either water or varying quantities of biotinylateddNTPs, and are cleaved by treatment with endonuclease V.

Dynabeads MyOne C1 magnetic streptavidin beads show no reduction in areain the presence of 10 or 100 pmol biotinylated ddNTP. However, a largedecrease in signal is seen with the addition of 500 pmol of biotinylatedddNTP.

Solulink magnetic beads show no reduction in signal in the presence ofup to and including 500 pmol of biotinylated dNTP. This indicates thatunincorporated biotinylated ddNTP from an extension reaction would notcause a decrease in final signal if it does not total greater than 500pmol.

These results in combination with experiments not outlined in thisreport indicate Solulink beads have a greater tolerance to biotinylatedsmall molecules inhibiting the binding of biotinylated extensionproduct. This is probably due to the greater binding capacity of thebeads, which is reported to be 2500 vs. 500 pmol biotinoligonucleotides/mg (FIG. 22 ).

Cleavage

The mass tags were cleaved from the extension product by addition of asolution containing 12 U Endonuclease V (NEB) and 10 mM MagnesiumAcetate (Sigma) and incubation at 37° C. for 4 hours in a Thermomixer R(Eppendorf) shaking at 1500 rpm. After incubation the magnetic beadswere pelleted using a magnetic rack and the supernatant was removed.

Effect of deoxyinosine position on cleavage properties: This experimentwas designed to analyze the ability of endonuclease V to cleave anextension product containing a deoxyinosine nucleotide in differentlocations. Four oligonucleotides were designed to simulate an extensionproduct (contained a 3′ biotin and a deoxyinosine nucleotide), whichonly differed in the location of the deoxyinosine nucleotide. Thedeoxyinosine was placed 10, 15, 20 and 25 base pairs from the 3′nucleotide containing the biotin moiety.

The mass tag signal seen after cleavage of the supernatant from thebinding step (unbound oligonucleotide) indicates a similar quantity ofoligonucleotide was bound onto the magnetic streptavidin beads for alloligonucleotides. However, after cleaving the oligonucleotides bound tothe magnetic streptavidin beads a clear pattern is seen. The larger thedistance of deoxyinosine to the 3′ end of the oligonucleotide thegreater the signal and presumably the cleavage. These results led todesign all extension oligonucleotides so the deoxyinosine is at least 20nucleotides from the putative 3′ end of the extension product (FIG. 23).

Bead and Endonuclease V titration: The quantity of Solulink magneticstreptavidin beads to efficiently capture biotinylated extensionproducts, and endonuclease V to cleave captured product to release masstags was evaluated in a series of experiments using 70 plex assays.

The initial experiment compared 10, 20 and 30 μl of Solulink magneticstreptavidin beads and 10, 20 and 30 units of endonuclease V.Signal-to-noise ratios show similar results with all combinations testedexcept when using 20 and 30 μl of magnetic beads in combination with 10units of endonuclease V (FIG. 24 ). Identical results were seen whencalling genotypes manually comparing 30 μl of beads and 30 Uendonuclease V with 10 μl of beads and 10 U endonuclease V.

To follow up these results an experiment compared the followingconditions; 10 μl beads/10 U endonuclease V; 5 μl beads/10 Uendonuclease V, 10 μl beads/5 U endonuclease V, and 5 μl beads/5 Uendonuclease V. When examining either manual genotype calls orsignal-to-noise ratio similar results were seen when using either 10 or5 μl of magnetic beads (FIG. 25 ). However, when using 5 U endonucleaseV there was a significant reduction in both manual calls andsignal-to-noise ratio when compared to 10 U endonuclease V.

To confirm these results an additional experiment compared the followingconditions; 10 μl beads/12 U endonuclease V; 5 μl beads/6 U endonucleaseV, 5 μl beads/12 U endonuclease V, and 5 μl beads/18 U endonuclease V.When comparing both manual genotype calls and signal-to-noise ratios,similar results were seen when comparing 10 or 5 μl of Solulink magneticbeads (FIG. 26 ). When comparing different quantities of endonuclease V,similar results were seen with 12 and 18 U endonuclease V. However, whenusing 6 U of endonuclease V a reduction in signal was observed (FIG. 26).

Example 11: Alternative Oligonucleotide Cleavage Mechanism

Ribonucleotide: Initial experiments used extension oligonucleotideswhich included a ribonucleotide. After extension and subsequent captureon magnetic streptavidin beads the mass tags are released by RNase Acleavage of the ribonucleotide. The method is outlined in the followingsection. The assays were developed for the SNPs rs1000586 and rs10131894in combination. The 2 plex reaction worked well and the genotypes areclearly seen (FIG. 8 ). A challenge to overcome in the future iscleavage of the ribonucleotides-containing oligonucleotides due tofreeze thawing.

Photocleavable: To explore an alternative to cleavage of deoxyinosinewith endonuclease V oligonucleotides containing a photocleavable linkerwere tested (IDT). The linker contains a 10-atom spacer arm which can becleaved with exposure to UV light in the 300-350 nm spectral range.

Methylphosphonate: As a further alternative to using cleavage ofdeoxyinosine with endonuclease V, oligonucleotides containing amethylphosphonate modification were examined. The oligonucleotidescontain a modification of the phosphate backbone at a single position,where oxygen is substituted with a methyl group. This results in aneutrally charged backbone which can be cleaved by Sodium hydroxide(NaOH), or potassium hydroxide (KOH) and heat. A series of experimentsshowed that the oligonucleotides can be cleaved by addition of as littleas 50 mM of NaOH or 200 mM KOH and heating at 70° C. for one hour.

dSpacer, Phosphorothioate/Phosphoramidite: Three alternative cleavagemechanisms that have not been explored in detail are the replacement ofa nucleotide with a 1′, 2′-Dideoxyribose (dSpacer) and the backbonemodifications creating either a phosphorothioate or phosphoramidite. Aphosphorothioate modification replaces a bridging oxygen with a sulphur.This enables the backbone to be cleaved with treatment with either 30/50mM aqueous sliver nitrate solution (with/without dithiothreitol) or 50mM iodine in aqueous acetone. A phosphoramidite modification replaces abridging oxygen with a amide group. The resulting P—N bond can becleaved with treatment with 80% CH₃COOH or during the MALDI-TOFprocedure.

Example 12: Isolation of Biotinylated Extension Products Using a BiotinCompetition Release Method

A method for purifying biotinylated extension products and releasing theproducts from streptavidin coated magnetic beads using free biotin isdescribed herein, and illustrated in FIGS. 27A-27G. In some embodiments,the method is utilized to purify and isolate and analyze single baseextension reactions for polymorphism identification

A genomic region of interest (e.g., a region having genetic variation)can be targeted using PCR based methods (see FIG. 27A). After PCRamplification of a region of interest, the reaction products weretreated with shrimp alkaline phosphatase (SAP) to dephosphorylateunincorporated nucleotide triphosphates. The region of interest,including a polymorphism of interest, can be targeted by nucleotideprobes using single base extension (SBE) reactions that correspond tothe nucleotide residue of interest (e.g., polymorphism; see FIG. 27B).The SBE reactions utilize biotin labeled dideoxynucleotide triphosphateterminators. Biotinylated extension products were subsequently captured(see FIG. 27C), washed and purified away from (see FIG. 27D) unusedreaction components utilizing streptavidin coated magnetic beads andmagnetic separation.

The purified extension products were subsequently eluted from thestreptavidin coated magnetic beads by competition with free biotin underelevated temperature conditions (see FIG. 27E). The eluant containingthe biotinylated extension products from the region of interest aredispensed (see FIG. 27F) onto a SpectroCHIP® (SEQUENOM) and analyzedusing MALDI-TOF mass spectrometry (see FIG. 27G). Reaction componentsand conditions are described herein.

Methods

Reaction components used in PCR amplification reactions.

Final Concentration Reagent per Reaction RNase-free ddH₂O N/A 10X Buffer1x dNTPs (25 mM each) 200 μM MgCl₂ (25 mM) 1.0 mM HotStar Taq (5 U/μl)N/A Primer Mix (1 μl M) 200 nM Template various

Reaction components used in Shrimp Alkaline Phosphatasedephosphorylation reactions.

Final Concentration Reagent per Reaction RNase-free ddH₂O N/A 10X SAPBuffer 0.24X SAP enzyme (1.7 U/ul) 0.073 U

Reaction components used in single base extension reactions.

Final Concentration Reagent per Reaction H₂O N/A 10X Buffer 0.222XPrimer mix (9 μM) 1 μM Biotinylated ddNTPs (250 μl μM) 5.56 μMThermosequenase (32 U/μl) 1.3 U

Binding and wash solutions used isolate and purify biotinylatedextension products.

Solution Composition 2X Binding 2M NaCl, 10 mM Tris pH 7.5, 1.0 mM EDTABuffer 10X WASH 100 mM Tris-HCl pH 8.0 Buffer 1X WASH 10 mM Tris pH 8.0Buffer

Components and procedure for preparing streptavidin coated magneticbeads.

Reagent Volume per Reaction [μl] Beads 10

Place on magnet at least 3 min to concentrate beads; remove supernatant.

Add 2× Binding Buffer to the tube as follows:

Volume per Reagent Reaction [μl] 2x Binding Buffer 10

Mix gently, then place 2-3 min on magnet to capture beads; removesupernatant.

Add 2× Binding Buffer to the tube to repeat for a total of 2 washes asfollows:

Volume per Reagent Reaction [μl] 2x Binding Buffer 10

Mix gently, then place 2-3 min on magnet to capture beads; removesupernatant.

Resuspend beads in 2× Binding Buffer as follows:

Volume per Reagent Reaction 25

Components and procedure for capturing biotinylated extension productsusing streptavidin coated magnetic beads.

-   -   Add an equal volume the 2× Binding Buffer with Beads to each        well of PCR reaction:    -   25 μl Beads in 2× Binding Buffer    -   25 μl Extension product

Rotate plate to mix for 15-30 min at room temperature.

Place plate on magnetic separator, remove supernatant.

Components and procedure for purification and washing of biotinylatedextension products using streptavidin coated magnetic beads.

-   -   Add 1× WASH Buffer as follows:

Volume per Reagent Reaction [μl] 1x WASH Buffer 50

-   -   Mix gently, then place 2-3 min on magnet to capture beads;        remove supernatant.    -   Add 1× WASH Buffer to the tube to repeat for a total of 2 washes        as follows:

Volume per Reagent Reaction [μl] 1x WASH Buffer 100

-   -   Mix gently, then place 2-3 min on magnet to capture beads;        remove supernatant.        -   Add WATER as follows:

Volume per Reagent Reaction [μl] WATER 100

-   -   Mix gently, then place 2-3 min on magnet to capture beads;        remove supernatant.    -   Add WATER to the tube to repeat for a total of 2 washes as        follows:

Volume per Reagent Reaction [μl] WATER 100

-   -   Mix gently, then place 2-3 min on magnet to capture beads;        remove supernatant.

Elution of captured biotinylated extension products from streptavidincoated magnetic beads for subsequent analysis.

The biotinylated extension products were eluted from the streptavidincoated magnetic beads using competition with free biotin at elevatedtemperatures. The reaction conditions are given in the table below.

-   -   Add 15 μl of BIOTIN (Resin treated, 25 ng/μl).    -   Heat to 90° C. for 5 min. then chill to 4° C.    -   Place on magnet for 2-3 min to capture beads.

After capturing the magnetic beads as described in the table, the eluantwas removed from the beads and prepared for further analysis. In someembodiments, preparation for further analysis includes dispensing orspotting onto a solid support suitable for use in MALDI-TOF massspectrometry. In certain embodiments, the solid support is aSpectroCHIP® (SEQUENOM) solid support. The biotinylated extensionproducts sometimes are analyzed by MALDI-TOF mass spectrometry, whichuses differences in mass of the extension products to elucidate thegenotype of the sample at the region of interest (e.g., at the site ofthe polymorphism). A representative spectrum tracing is shown in FIG. 28.

FIG. 28 illustrates the mass differences in single base reactionproducts analyzed by MALDI-TOF mass spectrometry. Unextended primer isshown on the spectrum tracing at a mass of approximately 5997 Daltons.Two extension reaction products also are shown in FIG. 28 , a ddUreaction product representing about 10% of the input template and havinga mass of approximately 6665 Daltons, and a ddA reaction productrepresenting about 90% of the input template and having a mass ofapproximately 6687 Daltons. The additional dotted lines at mass 6510 and6579 are the expected mass for alleles of the BRAF_2_WT and BRAF_2_Rmarker single base extension products.

Example 13: Extension and Releasing Extended Oligonucleotides from aSolid Phase

A typical multiplex (i.e. iPLEX) was followed up to the extension step(e.g. Example 8). PCR amplification of the region of interest wasperformed followed by SAP dephophorylation of the unincorporatednucleotides. The single base extension utilized biotinylateddideoxynucleotides. This extension was performed by either includingonly the nucleotides corresponding to the minor species or all fournucleotides. Biotinylated oligonucleotides were captured usingstreptavidin beads from different manufacturers. For the releasing step,the inosine cleavage method was compared to using free-biotin to competeoff bound biotinylated oligonucleotides. Other components of thisreaction were similar to assays previously described (e.g. Example 8).Conditions used for the PCR amplification, SAP dephosphorylation andextension are shown in Table 8 below.

TABLE 8 PCR Setup Final Concentration Volume for Single Reagent perReaction Reaction (ul) RNase-free ddH₂O N/A 0.16 10X Buffer 1x 0.50dNTPs (25 mM each) 200 uM 0.04 MgCl₂ (25 mM)  1.0 mM 0.20 HotStarTaq (5U/ul) N/A 0.10 Primer Mix (1 uM) 200 nM 1.00 Competitor Titration 3.00TOTAL 5.00 PCR Amplification Parameters 95 C. 15 min 95 C. 20 sec 10cycles 56 C. 20 sec 72 C.  1 min 72 C.  3 min  4 C. ∞ SAP Setup FinalConcentration Volume for Single Reagent per Reaction Reaction (ul)RNase-free ddH₂O N/A 1.53 10X SAP Buffer 0.24X 0.17 SAP enzyme (1.7U/ul) 0.073 U/ul 0.30 TOTAL 2.00 SAP Incubation 37° C. for 20 minutes85° C. for 10 minutes  4° C. forever Extension Setup Final ConcentrationVolume for Single Reagent per Reaction Reaction (ml) H₂O N/A 0.56 10XBuffer 0.222X 0.20 Primer Mix (9 uM)   1 uM 1.00 Biotinylated ddT&A (250uM) 5.56 uM 0.20 Thermosequenase (32 U/ul)  1.3 U 0.04 TOTAL 2.00Extension Parameters 94° C. for 30 seconds 94° C. for 5 seconds 40cycles 52° C. for 5 seconds 5 cycles 80° C. for 5 seconds 72° C. for 3minutes  4° C. forever

After extension, the reaction was introduced to streptavidin coatedmagnetic beads. The extended products were allowed to capture for ashort duration. Subsequent wash steps were conducted to remove reactioncomponents except the captured extension products (Table 9). Thisprocedure removed salts that produce interfering adducts in MALDI-TOFmass spectrometry, which allowed for the removal of the anion exchangeresin procedure now employed with current iPLEX workflow. After washing,captured extension products were eluted from the beads by introducing ahigh molar free biotin solution at 25 ng/ul. After elution, theextension products remained in the eluent. The cleaned analyte was nowready for dispensing on the bioarray chip and was substantially freefrom unextended primer, salts, and other contaminants that can obscure alow abundant species on the MALDI-TOF spectra, thus allowing for a moresensitive detection. FIGS. 29A-29G depict a flow chart of thisprocedure.

TABLE 9 Bead Conditioning Vortex MyOne Steptavidin C1 beads tocompletely resuspend beads. Transfer beads to a tube as follows: ReagentVol per Reaction Beads 10 Place on magnet at least 3 min to concentratebeads; remove supernatant. Add 2x Binding Buffer to the tube as follows:Reagent Vol per Reaction 2x Binding Buffer 10 Mix gently, then place 2-3min on magnet to capture beads. Add 2x Binding Buffer to the tube torepeat for a total of 2 washes as follows: Reagent Vol per Reaction 2xBinding Buffer 10 Mix gently, then place 2-3 min on magnet to capturebeads; remove supernatant. Capture Resuspend beads in 2x Binding Bufferas follows: Reagent Vol per Reaction 2x Binding Buffer 25 Add an equalvolume the 2x Binding Buffer with Beads to each well of PCR reaction: 25ul Beads in 2x Binding Buffer 25 ul PCR product 50 ul Rotate plate tomix for 15-30 min at room temp. Place plate on magnetic separator,remove supernatant. Bead Wash Add 1x WASH Buffer as follows: Reagent Volper Reaction 1x WASH Buffer 50 Mix gently, then place 2-3 min on magnetto capture beads; remove supernatant. Add 1x WASH Buffer to the tube torepeat for a total of 2 washes as follows: Reagent Vol per Reaction 1xWASH Buffer 100 Mix gently, then place 2-3 min on magnet to capturebeads; remove supernatant. Add WATER as follows: Reagent Vol perReaction WATER 100 Mix gently, then place 2-3 min on magnet to capturebeads; remove supernatant. Add WATER to the tube to repeat for a totalof 2 washes as follows: Reagent Vol per Reaction WATER 100 Mix gently,then place 2-3 min on magnet to capture beads; remove supernatant.Elution Add 15 of BIOTIN (Resin treated, 25 ng/ul). Heat to 90 C. for 5min. then chill to 4 C. Place on magnet for 2-3 min to capture beads.Remove supernatant to a clean 96-well plate and run on MALDI.

Inosine Cleavage

As an alternate elution method, inosine cleavage of mass tags from thecaptured extension products was performed. This method deviated from thebiotin competition approach for extension oligonucleotide design. Thisapproach required a non-templated sequence on the 5′ end of theextension primer to correspond to the extension product. In addition,this oligonucleotide was synthesized with an inosine residue thatseparates the complementary sequence of the target and the mass tagsequence identifier. Through this inosine residue, the mass tag wascleaved from the capture agent by endonuclease V activity. The cleavageportion of this process is illustrated in FIGS. 30A and 30B.

Streptavidin Bead Selection and Elution Evaluation (Biotin Vs InosineCleavage)

Five streptavidin bead products were selected for evaluation. Theselection was based on surface characteristics and binding capacity offree biotin. These characteristics are listed in Table 10.

TABLE 10 Bead characteristics Binding capacity of free Bead Surfacebiotin (pmoles/mg beads) Dynal M270 Hydrophilic 650-1350 Dynal M280Hydrophobic 650-900  Dynal C1 Hydrophilic >2,500 Dynal T1Hydrophobic >1,300 Solulink Hydrophilic >1,300

The performance of the beads was evaluated using oligonucleotidessynthesized with 3′ biotin. Two oligonucleotides were designed with anidentical “sequence specific” region; one had no 5′ modification whilethe other contained a 5′ mass tag with an inosine residue. To evaluateefficiency of capture and elution strategy, a comparative measure withineach spectrum was employed. Additional oligonucleotides were designed sotheir respective size was within an acceptable mass range of the elutedproducts for comparison.

The testing procedure included capturing the 3′ biotinylatedoligonucleotides, performing a set of washes, and subsequent elution(biotin competition vs. inosine cleavage). This strategy alleviatesvariability that may have been introduced during the PCR and extensionsteps. Beads and elution strategies were evaluated by response tolimiting the capturable oligonucleotide. This evaluation was performedby serially diluting the 3′ biotinylated oligonucleotide from 2 uM to0.031 uM. For each concentration tested, an equal amount ofquantification oligonucleotide was added to the eluent to measure beadcapture and elution efficiency.

The ratio of capture oligonucleotide to quantification oligonucleotideheights was measured at each concentration for each bead evaluated. Thisinitial experiment showed the biotin competition method to outperformthe elution method. Both elution methods exhibited a captured product atthe lowest starting input, but the biotin capture clearly showed morecaptured and eluted product, as displayed by the ratio. This outcome wasevident regardless of bead used and the biotin competition was chosen asthe elution strategy. The data also showed underperformance of DynalM280and DynalT1. The data reflecting this experiment can be seen in FIG. 31and FIGS. 32A and 32B.

Bead Evaluation

The next approach was to analyze capture beads for further development.This experimentation utilized all steps of the proposed ultra-sensitivedetection workflow to evaluate bead performance from an extended productoff a PCR template. In order to control input material, competitoroligonucleotides were used as template material. PCR, SAP, extension andcapture were performed as outlined previously. The template complement,Biotin-ddUTP, was the sole nucleotide used in the extension reaction.Competitor oligonucleotide template concentration was serial dilutedfrom approx. 60,000 molecules to approx. 30 molecules. Each dilution wasreplicated six times. For all reactions 1 ul of a 1 uM solution ofextension oligonucleotides was used. The beads evaluated were DynalM270, Dynal C1, and Solulink. To elucidate binding performance of eachbead using the same strategy as employed in the previous study, 1 ul ofa 1 uM quantification oligonucleotide solution was added to the eluentpost biotin capture.

The results of this evaluation demonstrated Dynal C1 to outperform bothSolulink and M270 at all template concentrations. M270 failed to captureany product. For Dynal C1, a gradual decline in the ratio of extensionproduct to quantification oligonucleotide using either height or area asthe measure was observed as it related to input amount. This samerelationship was not observed with Solulink, suggesting a limit ofcapture. The data for this experiment can be seen in FIG. 33 and FIG. 34.

Genomic Variants (i.e. Genomic Mix Model)

With the essential components and procedures for the processestablished, development proceeded to actual samples. A model system wasdeveloped using samples and assays well characterized in a previousvalidation (Oncocarta validation). The genetic material used iscommercially available from ATCC and is known to carry somaticmutations. Sample HTB-26D (genomic DNA of a cell line derived frombreast adenomcarcinoma) carries a mutation in theserine/threonine-protein kinase B-Raf (BRAF) encoding region.Specifically, this sample has previously shown a somatic mutation inBRAF-2 (Wild type—G; Mutant—T). This sample was characterized as being30% mutant. Sample HTB-38D (genomic DNA of cell line derived fromcolorectal adenocarcinoma) also carries a mutation in the BRAF region.This sample has previously shown a mutation in BRAF-15 (Wild type—T;Mutant—A). This sample was characterized as 15% mutant. HTB-26D is wildtype for BRAF-15 and HTB-38D is wild type for BRAF-2.

One rationale for the selection of these assays and samples, beyond theease of obtaining genetic material, was the specific genotypes involved.Biotin-ddCTP and biotin-ddTTP are only separated by 1 Da in mass.Although this mass difference is within the resolution of the MALDI-TOFinstruments, a larger mass difference between products was evaluated.Subsequently, a different vendor was found to offer biotin-ddUTP with a16 carbon linker (vs. the 11 carbon linker of the original set).Replacement of the 11 carbon linker with the 16 carbon linker in thisassay alleviated any potential design issues.

To evaluate sensitivity in this model system, the two samples were mixedto further dilute each corresponding somatic mutation. The two sampleswere mixed at different ratios, titrating the somatic mutation in BRAF-2for sample 26D from 30% to 1.5% and titrating the BRAF-15 variant from15% to 0.75% for sample 38D. Each titration point was run in duplicateand recombined after SAP. The mixed analyte was redistributed to twodifferent reactions. One of the reactions was subject to all fourbiotin-ddNTP's while the other used just biotin-ddUTP (for BRAF-2) andbiotin-ddATP (for BRAF-15). Capture and elution were performed with theDynal-C1 beads and free-biotin competition. FIG. 35 shows the resultsfor these four scenarios.

The BRAF-2 mutant showed a slight signal in the reaction run with all 4biotin-ddNTP's (top left panel, FIG. 35 ). Ordinarily this sort ofsignal would not be considered significant above baseline noise. Withjust biotin-ddUTP (for BRAF-2 reaction, bottom left of FIG. 35 ) a veryclear and distinct signal was observed for the mutant. This sameobservation held true for the 0.75% mutant in the BRAF-15 assay forsample 38D. A very clear and distinct signal was observed for theBRAF-15 mutant when just biotin-ddATP (bottom right of FIG. 35 ) wasincluded in the extension composition. This data demonstrated asignificant increase in the signal to noise ratio (SNR) by excluding thebiotin-ddNTP's corresponding to the more abundant wild type sequence. Inthe case of BRAF-15, there was no observed signal for the mutant in thereaction when all 4 biotin-ddNTP's were included in the extensionreaction (top right panel, FIG. 35 ).

Detection and Quantitation of a low Abandance variant

In some embodiments the amount of molecules of a target mutant variant(e.g. low abundant variant) present in an assay where the wild type(e.g. high abundance species) extension product is not generated isdetermined by the use of a synthetic template included in the extensionreaction. The initial goal of this evaluation was to assess the abilityto reliably detect the minor contribution (i.e. of a low abundancemutant) in a mixture at sensitive levels. The post-PCR enrichmentstrategy summarized here defines this ability is possible by effectivelyremoving the wild type (e.g. high abundance species) extension product.It is possible to determine the amount (e.g. copy number, concentration,percentage) of target mutant molecules present (i.e. mutant extensionproducts) in an assay (e.g. extension reaction), if the input quantityof template is known. The amount of target (e.g. copy number,concentration, percentage) mutant variant (i.e. mutant extensionproducts) and/or percentage of target mutant variant in the sample isquantified by including a known amount of synthetic template in theextension reaction. The synthetic template can hybridize to anoligonucleotide species and contain a base substitution at the mutantposition located just 3′ of the oligonucleotide species to be extended.The base substitution is different than the wild type or target mutantvariant (e.g. first variant, low abundant variant, SNP). The basesubstitution present in the synthetic template is not present in thesample prior to introduction of the synthetic template. A ddNTP that iscomplementary to the base substitution in the synthetic template is alsointroduced into the reaction. Oligonucleotide species that hybridize tothe target mutant variant are co-amplified (e.g. co-extended) witholigonucleotide species that hybridize to the synthetic template. Byperforming multiple reactions, that include serial dilutions of asynthetic template, the amount and/or percentage of the target mutantvariant can be ascertained. The amount and/or percentage of the targetmutant variant is determined by the amount of synthetic template thatyields equal extension product as the target mutant variant.

Mutant quantification, as described, was carried out on a genomic mixmodel. With constant mutant percentages of 5, 1, 0.5 and 0.1, synthetictemplate titrations were applied targeting a theoretical number ofmolecules given a total input DNA of 20 ng. The result showed anaccurate count of mutant molecules for the 5 and 1% samples. The processwas not as accurate at lower levels, presumably due to PCR sampling biaswith limited template. FIG. 36 shows the titration profile for the 1%mutant.

CONCLUSIONS

Elimination of the wild type extension product can increase sensitivityof the multiplex assay disclosed herein. In some embodiments, assays ofthe same plex have the same wild type genotype, or have the same mutantgenotype. In some embodiments, synthetic templates or plasmid constructswith designed “mutations” against a genomic DNA of a healthy population(i.e. HAPMAP consortium samples) can be used. This strategy canalleviate design concerns and can have the distinct advantage ofartificially creating different mutant percentages in different assaysfor the same plex (competitor only). In some embodiments, synthetictemplates (e.g. plasmids or oligonucleotide templates) are used ascontrols. In some embodiments, synthetic templates (e.g. plasmids oroligonucleotide templates) are included in kits.

In some embodiments, designs are tailored toward the wild type (i.e moreabundant) nucleotide. In this way all assays in one plex share the samenucleotide for wild type and the extension mix used leaves out thisnucleotide. In some embodiments, designs can be tailored toward themutant. In some embodiments, all assays in a plex have the same mutantbase in common. In certain embodiments, the extension mix only containsthe mutant base. In some embodiments, there is a risk of non-specificinteraction with the overwhelming background wild type DNA. In someembodiments there should be at least one control plex representing eachwild type base removed, or each mutant base left in depending on thedesign style chosen.

Workflow Improvements

The processes as described are amenable to automation. Key steps toconsider for automation can be bead conditioning, bead addition, beadwashing, and aspiration of the eluted product.

Example 14: Detecting “Wild Type” and “Mutant” Genotypes Using MultiplexAssays and Kits

Assay Design

A hindrance to a more sensitive detection has been the presence of thewild type peak scaling the intensity to a point where low level mutantsare no longer visible above baseline. Removing the wild type peak fromdetection has improved sensitivity and signal to noise ratio. Assaysdesigned within a single plex have the same wild type peak in commonwith corresponding wild type base removed from the extension reaction,or designed to have the same mutant peak in common with only thatspecific base included in the extension reaction (Table 11). In terms ofmaterial cost, the strategy of choosing a plex directed toward themutant allele with only one base used in the extension reaction is usedfor a model system.

Multiplex (i.e. Plex) designs are divided into three classes of assays.The three classes represent each of the other 3 nucleotides as the wildtype. Four multiplex assays (i.e. plexes) are designed using thisrationale with each plex targeting a different mutant base. This assaydesign strategy allows the exploration of all possible wild type/mutantcombinations.

The design incorporates six regions from a Lung Panel. Each designed“mutant” is interrogated in the forward and reverse direction tofacilitate the requirement of all possible wild type/mutantcombinations. The design avoids overlap from extension oligonucleotideand PCR primer so as to avoid any potential exonuclease derivedadditional signals. The mutation designed into the model representsactual somatic mutations used in the Lung Panel. The design withmultiplex is outlined in Table 12. The four multiplexes are designed sothey can also be multiplexed together in one plex using acyclicextension mix. The design incorporates an EcoRI site separating theregions from each other (FIG. 37 ). Prior to using the model, theplasmid is cleaved through EcoRI restriction digest to separate theregions and more adequately reflect a genomic context.

TABLE 11 Extension nucleotide mix Design with all assays sharing thesame wild type nucleotide in a plex Plex with wild type-T C, G, A Plexwith wild type-C T, G, A Plex with wild type-G T, C, A Plex with wildtype-A T, C, G Design with all assays sharing the same mutant nucleotidein a plex Plex with mutant-T T Plex with mutant-C C Plex with mutant-G GPlex with mutant-A A

TABLE 12 Multiplex Region Wild Type/Mutation Extension Direction MutantC DDR2_L63V G/C R Multiplex EPHA3_N379R A/C R JAK2_Y931C T/C RAlubmin_Ctrl_C A/C R Mutant A NOTCH1_R2328W G/A R Multiplex TP53_R249WT/A F ALK_C4453A C/A F Alubmin_Ctrl_A G/A F Mutant T ALK_C4493A G/T RMultiplex NOTCH1_R2328W C/T F TP53_R245W A/T R Alubmin_Ctrl_T C/T RMutant G EPHA3_N379K T/G F Multiplex DDR2_L63V C/G F JAK2_Y931C A/G FAlubmin_Ctrl_G T/G F

Controls

Elements of the process lead to controls for downstream analysis.Failure to capture a product can be due to limited template, failedPCR/extension, or failed capture and elution. To evaluate issues withthese specific variables, a control assay is included in each plexdesigned. The control designed targets the human albumin gene. A controlis represented in each reaction that is sufficiently templated withproper functioning PCR and extension. Four separate extensionoligonucleotides are intended for this control. These extensionoligonucleotides target a residue representing each of the fournucleotides and are used with the appropriate extension nucleotide mix.

Subsequent to the extension assay, a 5′ biotinylated oligonucleotidewith 3′ inverted dTTP is spiked into all assays as a control for captureand elution. The absence of this signal coinciding with an absence ofany analyte informs the user of a failed capture and/or elution. Themolarity of this particular control should not overwhelm the reaction toavoid masking any low level mutant in detection.

Elution Optimization

Captured and washed products are eluted into 15 ul of high molar biotinsolution. New hardware which pellets beads at the appropriate height for15 ul elution is optimized for the process using Matrix PlateMate 2×2and/or the Epimotion 5075. A titration experiment is used to evaluatethe performance of the new plate. As a test of the process, thisevaluation is done in triplicate for each dilution of capture control.The capture control is spiked into an iPLEX simulant solution. The testsolution undergoes the typical post-extension process using thehardware.

Automation adjustments for the PlateMate are made prior to elutionexperimentation. The titration encompasses twelve steps of a serialdilution bringing input capture oligonucleotide from 2500 molecules toapproximately 1 molecule. The method with new hardware is optimized forthe ability to maintain a pellet during the washes without loss ofbeads. Ultimately, the performance is judged by the sensitivity asdemonstrated by detection of capture oligonucleotide.

Quality Control

The four multiplexes are initially run with the typical iPLEX processusing an acyclic extension mix on plasmid alone. This is performed as aquality measure of plasmid manufacturing. Restriction digest is alsooptimized and visualized in an agarose gel to ensure complete digestioninto constituent fragments.

Capture Control Optimization

There is an initial experiment to determine what concentration ofcapture control is appropriate for subsequent experiments. This controlis useful for those reactions where there is no mutant and therefore noextension peak. In this situation, it is necessary to determine if theabsence of a peak is the result of the absence of sufficient template togenerate an extension product or a failure of capture or elution. Atitration of plasmid is performed in quadruplicate. For each replicate,a different concentration of capture oligonucleotide is used. Thetitration of plasmid mirrors the sensitivity experiment with eightdilutions from 50% mutant to 0.01% mutant and a no mutant reaction. Thelowest concentration of capture control used is determined from theelution optimization experiment. This concentration is used as input forone replicate and doubling concentrations for the other threereplicates. Performance of the capture control is evaluated by howmutant assay detection is effected by the presence of a capture controlpeak. The peak should not obscure low level mutant by being tooprevalent in the spectra. However, the capture control peak must beclearly detected at low level mutant concentrations. The findings ofthis experiment are the basis for capture control concentration insubsequent sensitivity, specificity and concordance assays. The 3′inverted dT is not necessary for these reactions, as the oligonucleotideis not involved in the extension reaction. However, the control isdesigned this way as to allow incorporation of the control in theextension reaction itself.

Sensitivity and Specificity

Establishing a sensitivity threshold of the process involves titratingplasmid DNA relative to the human DNA. Sensitivity thresholds aredetermined when no mutant analyte is detectable or to a point were asingle copy of the variant is used for the minority template. Variousdilutions of the mixture are used. The number of template mutantmolecules is 15000, 3750, 938, 235, 59, 15, 4 and 0. The respectivenumber of wild type copies is 15000, 26250, 29062, 29765, 29941, 29985,29996 and 30000. Combined, these eight mixtures represent a 50%, 12.5%,3.13%, 0.78%, 0.2%, 0.05%, 0.01% and 0% mutant concentration,respectively. Total template is 90 ng/rxn, or 30,000 genomic copies.Every dilution of each plex is run with 48 replicates. An extra twoplates running 48 non-templated reactions for each multiplex is run as acontrol to assess the extent of non-specific interactions. Additionally,a “golden standard” plate of 48 samples run in duplicate using the 50%mutant and 12.5% mutant establishs proper ratios are being employed. Intotal, the sensitivity and specificity trial requires nineteen 96 wellplates. PCR and SAP is implemented according to current iPLEX protocol.Post-SAP reactions are subject to an extension reaction containingbiotin-ddNTP's as the alternative terminating nucleotide substrate(except the “golden standard plate”). All other reaction components willremain the same. Table 13 shows the model system dilution setup in termsof molecules number and weight of each constituent DNA. Table 14 andTable 15 lists the entire process from PCR to extension inconcentrations for each component on a per reaction basis.

Initial analysis considers at what point no signal is observed toestablish a statistically significant sensitivity threshold. Thisanalysis considers the overall data encompassing all multiplexes, andalso takes into account variability that may occur when extending aspecific base as well as what impact, if any, the wild type backgroundgenotype has on successful extension. This analysis evaluates how thesensitivity has effect on specificity.

TABLE 13 Extension mix table Total Weight Weight Weight Total % (ng)molecules (ng) molecules (ng) molecules Mutant 45 15000 45 15000 9030000 50 78.75 26250 11.25 3750 90 30000 12.5 87.19 29062 2.81 938 9030000 3.13 89.3 29765 0.71 235 90 30000 0.78 89.82 29941 0.18 59 9030000 0.2 89.96 29985 0.05 15 90 30000 0.05 89.99 29996 0.01 4 90 300000.01 90 30000 0 0 90 30000 0

Concordance

Concordance analysis considers the data collected from the sensitivityand specificity experiments. All replicates are gauged for agreementwithin each experiment as well as agreement across experiments. The“golden standard genotype” is established by running the model systemitself in the model system quality control.

An additional measure of concordance is performed with samples providedby Horizon Diagnostics. Horizon Diagnostics provides genetically definedgDNA and FFPE cell reference standards. Evaluation considers no morethan 23 samples that are FFPE prepared. Eight to fifteen mutations areselected from a list prepared by Horizon. To explore the power of thedetection system, mutants selected are purchased with the correspondingwild type version, or mixed with in house healthy population samples.The samples provided by Horizon are in 50% mutant state and a dilutionis required to evaluate sensitive detection in this context. Thedilution series is the same as utilized in the sensitivity andspecificity evaluation. In addition, a non-templated control is run tobring total sample number to 24. All samples are run in quadruplicatefor a total of eight 96 well plate. This experimental design willinclude an iPLEX “golden standard” using the Horizon Diagnostic sampleswithout dilution. This evaluation not only further assesses concordanceto traditional iPLEX, but also gives information on performance ofactual FFPE samples.

Control Variables

Pre-enrichment processing: All reactions are carried out according toiPLEX SOP up to the extension step. These processes are detailed inTable 14 and Table 15. All reagents used are controlled so that the samelot of reagents is used across the studies.

Bead Processing: Conditioning, wash, and elution steps have beenestablished from other studies to produce a reliable system. Afterelution strategies have been decided upon from the Pre-Testing phase, adefined protocol is used for all ensuing experiments.

Sample DNA: DNA derived from three sources is used for all studies. Thefirst is plasmid DNA containing the model system. Secondly is HapMapsamples from Utah residents of European ancestry. Lastly is DNA providedby Horizon Diagnostics.

Instrumentation: Pre and Post-PCR instrumentation include either thePlateMate 2×2 and/or the Hamilton Micro Lab 4000. Nanodispensing isperformed on the SEQUENOM Nanodispenser RS1000 with analyte detectionusing the MassARRAY Analyzer 4. All instrumentation serial numbers arecataloged.

TABLE 14 PCR Setup Final Concentration Volume for Single Reagent perReaction Reaction (ul) RNase-free ddH₂O N/A 1.16 10X Buffer (w/20 mMMgCl₂) 1x 0.50 dNTPs (25 mM each) 200 uM 0.04 MgCl₂ (25 mM)  1.0 mM 0.20Fastart Taq (5 U/ul)  0.1 U/rxn 0.10 Primer Mix (1 uM) 200 nM 1.00 DNA(5 ng/ul)  10 ng/rxn 2.00 TOTAL 5.00 PCR Amplification Parameters 95 C.15 min 95 C. 20 sec 45 cycles 56 C. 20 sec 72 C.  1 min 72 C.  3 min  4C. ∞ SAP Addition Final Concentration Volume for Single Reagent perReaction Reaction (ul) RNase-free ddH₂O N/A 1.53 10X SAP Buffer 0.24X0.17 SAP enzyme (1.7 U/ul) 0.073 U/ul 0.30 TOTAL 2.00 SAP incubation 37°C. 20 min 85° C.  5 min  4° C. ∞

TABLE 15 Extension Reaction Final Concentration Volume for SingleReagent per Reaction Reaction (ml) H₂O N/A 0.56 10X Buffer 0.222X 0.20Primer Mix (9 uM)   1 uM 1.00 Biotinylated ddNTPs (250 uM) 5.56 uM 0.20Thermosequenase (32 U/ul)  1.3 U/rxn 0.04 TOTAL 2.00 ExtensionParameters 94° C. 30 sec 94° C.  5 sec 40 cycles 52° C.  5 sec 5 cycles80° C.  5 sec 72° C.  3 min  4° C. ∞

Response Variables

The experiments employed in this test plan are evaluated for severalparameters. Control variables have potential impact on severalparameters. The ability of the process to deliver a reliable anddesirable result is evaluated. Any bearing on response variable that canbe reliably ascertained by control variables is accounted for.

-   -   Peak heights    -   Peak confidence scores    -   Expected genotypes

TABLE 16 Bead Conditioning Vortex MyOne Steptavidin C1 beads tocompletely resuspend beads. Transfer beads to a tube as follows: ReagentVol per Reaction Beads 10 Place on magnet at least 3 min to concentratebeads; remove supernatant. Add 2x Binding Buffer to the tube as follows:Reagent Vol per Reaction 2x Binding Buffer 10 Mix gently, then place 2-3min on magnet to capture beads. Add 2x Binding Buffer to the tube torepeat for a total of 2 washes as follows: Reagent Vol per Reaction 2xBinding Buffer 10 Mix gently, then place 2-3 min on magnet to capturebeads; remove supernatant. Capture Resuspend beads in 2x Binding Bufferas follows: Reagent Vol per Reaction 2x Binding Buffer 25 Add an equalvolume the 2x Binding Buffer with Beads to each well of PCR reaction: 25ul Beads in 2x Binding Buffer 25 ul PCR product 50 ul Rotate plate tomix for 15-30 min at room temp. Place plate on magnetic separator,remove supernatant. Bead Wash Add 1x WASH Buffer as follows: Reagent Volper Reaction 1x WASH Buffer 50 Mix gently, then place 2-3 min on magnetto capture beads; remove supernatant. Add 1x WASH Buffer to the tube torepeat for a total of 2 washes as follows: Reagent Vol per Reaction 1xWASH Buffer 100 Mix gently, then place 2-3 min on magnet to capturebeads; remove supernatant. Add WATER as follows: Reagent Vol perReaction WATER 100 Mix gently, then place 2-3 min on magnet to capturebeads; remove supernatant. Add WATER to the tube to repeat for a totalof 2 washes as follows: Reagent Vol per Reaction WATER 100 Mix gently,then place 2-3 min on magnet to capture beads; remove supernatant.Elution Add 15 of BIOTIN (Resin treated, 25 ng/ul). Heat to 90 C. for 5min. then chill to 4 C. Place on magnet for 2-3 min to capture beads.Remove supernatant to a clean 96-well plate and run on MALDI.

Bead Process

Beads are conditioned in in 2× binding buffer and a final volume of 25ul conditioned beads are added to the 9 ul extension reaction. Water isadded to bring the total volume to 50 ul. Bead capture is executed in a96 well plate to accommodate the volume. Capture of extension productsis performed on the hematology rotator at room temperature for 30minutes. After capture, the beads are washed of reaction components in a1× Tris buffer solution. This wash is repeated for a total of twowashes. The beads are then washed with water. The water wash is alsorepeated for a total of two more washes. Each wash step utilizes 100 ultotal volume. A 96 well plate magnet is used to pellet beads. The washsteps can be done manually or through the use of automation. Washedbeads are re-suspended in 15 ul of concentrated free biotin solution (25ng/ul; resin treated). Free biotin is allowed to out compete thebiotinylated extension products at 90° C. for 5 min. Of the 15 ulre-suspension, 10 ul is aspirated while beads are pelleted under magnet.This 10 ul clean eluent is dispensed into a 384 well plate fordispensing. Bead conditioning and washing parameters are shown in Table16.

Dispensing parameters require some alterations to typical dispensingprotocols given volume height and analyte characteristics. Aspirationoffset is set to 8 mm and dispense speed is changed to approximately 150mm/sec or other higher dispense speed to account for viscositydifference in this analyte from typical iPLEX biochemistry.

Example 15: Non-Limiting Examples of Embodiments

Provided hereafter are non-limiting examples of certain embodiments ofthe technology.

A1. A method for determining the presence or absence of a plurality oftarget nucleic acids in a composition, which comprises:

-   -   (a) preparing amplicons of the target nucleic acids by        amplifying the target nucleic acids, or portions thereof, under        amplification conditions;    -   (b) contacting the amplicons in solution with a set of        oligonucleotides under hybridization conditions, where each        oligonucleotide in the set includes a hybridization sequence        capable of specifically hybridizing to one amplicon under the        hybridization conditions when the amplicon is present in the        solution;    -   (c) generating extended oligonucleotides that include a capture        agent by extending oligonucleotides hybridized to the amplicons        by one or more nucleotides, wherein one of the one of more        nucleotides is a terminating nucleotide and one or more of the        nucleotides added to the oligonucleotides includes the capture        agent;    -   (d) contacting the extended oligonucleotides with a solid phase        under conditions in which the capture agent interacts with the        solid phase;    -   (e) releasing the extended oligonucleotides that have interacted        with the solid phase by competition with a competitor; and    -   (f) detecting the extended oligonucleotides released in (e) by        mass spectrometry; whereby the presence or absence of each        target nucleic acid is determined by the presence or absence of        the corresponding extended oligonucleotide.

A1.1. The method of embodiment A1, wherein (i) the mass of oneoligonucleotide species detectably differs from the masses of the otheroligonucleotide species in the set; and (ii) each oligonucleotidespecies specifically corresponds to a specific amplicon and therebyspecifically corresponds to a specific target nucleic acid.

A1.2. A method for determining the presence or absence of a plurality oftarget nucleic acids in a composition, which comprises:

-   -   (a) preparing amplicons of the target nucleic acids by        amplifying the target nucleic acids, or portions thereof, under        amplification conditions;    -   (b) contacting the amplicons in solution with a set of        oligonucleotides under hybridization conditions, wherein:        -   (i) each oligonucleotide in the set comprises a            hybridization sequence capable of specifically hybridizing            to one amplicon under the hybridization conditions when the            amplicon is present in the solution,        -   (ii) each oligonucleotide in the set comprises a mass            distinguishable tag located 5′ of the hybridization            sequence,        -   (iii) the mass of the mass distinguishable tag of one            oligonucleotide detectably differs from the masses of mass            distinguishable tags of the other oligonucleotides in the            set; and        -   (iv) each mass distinguishable tag specifically corresponds            to a specific amplicon and thereby specifically corresponds            to a specific target nucleic acid;    -   (c) generating extended oligonucleotides that comprise a capture        agent by extending oligonucleotides hybridized to the amplicons        by one or more nucleotides, wherein one of the one of more        nucleotides is a terminating nucleotide and one or more of the        nucleotides added to the oligonucleotides comprises the capture        agent;    -   (d) contacting the extended oligonucleotides with a solid phase        under conditions in which the capture agent interacts with the        solid phase;    -   (e) releasing the mass distinguishable tags in association with        the extended oligonucleotides that have interacted with the        solid phase from the solid phase by competition with a        competitor; and    -   (f) detecting the mass distinguishable tags released in (e) by        mass spectrometry; whereby the presence or absence of each        target nucleic acid is determined by the presence or absence of        the corresponding mass distinguishable tag.

A2. The method of any one of embodiments A1 to A1.2, wherein competitionwith a competitor comprises contacting the solid phase with acompetitor.

A3. The method of any one of embodiments A1 to A2, wherein thecompetitor consists of free capture agent, or a competing fragment ormultimer thereof.

A3.1. The method of embodiment A3, wherein the competitor consists offree capture agent.

A4. The method of any one of embodiments A1 to A3.1, wherein thenucleotide that comprises the capture agent is a capture agentconjugated to a nucleotide triphosphate.

A5. The method of embodiment A4, wherein the nucleotide triphosphate isa dideoxynucleotide triphosphate.

A6. The method of any one of embodiments A1 to A5, wherein the captureagent comprises a member of a binding pair.

A7. The method of any one of embodiments A1 to A6, wherein the captureagent comprises biotin.

A8. The method of embodiment A7, wherein the solid phase comprisesavidin or streptavidin.

A9. The method of any one of embodiments A1 to A6, wherein the captureagent comprises avidin or streptavidin.

A10. The method of embodiment A9, wherein the solid phase comprisesbiotin.

A11. The method of any one of embodiments A1 to A10, wherein releasingthe mass distinguishable tags by competition with free capture agent iscarried out under elevated temperature conditions.

A12. The method of embodiment A11, wherein the elevated temperatureconditions comprise treatment for about 5 minutes at about 90 degreesCelsius.

A13. The method of any one of embodiments A1 to A12, wherein (c) iscarried out in one container and the method further comprisestransferring the released mass distinguishable tags to another containerbetween (e) and (f).

A14. The method of any one of embodiments A1 to A13, wherein thesolution containing amplicons produced in (a) is treated with an agentthat removes terminal phosphates from any nucleotides not incorporatedinto the amplicons.

A15. The method of any one of embodiments A1 to A14, wherein theterminal phosphate is removed by contacting the solution with aphosphatase.

A16. The method of embodiment A15, wherein the phosphatase is alkalinephosphatase.

A17. The method of embodiment A16, wherein the alkaline phosphatase isshrimp alkaline phosphatase.

A18. The method of any one of embodiments A1 to A17, wherein theterminal nucleotides in the extended oligonucleotides comprise thecapture agent.

A19. The method of any one of embodiments A1 to A18, wherein one or morenon-terminal nucleotides in the extended oligonucleotides comprise thecapture agent.

A20. The method of any one of embodiments A1 to A19, wherein thehybridization sequence is about 5 to about 200 nucleotides in length.

A21. The method of any one of embodiments A1 to A20, wherein the solidphase is selected from a flat surface, a bead, a silicon chip, orcombinations of the foregoing.

A22. The method of any one of embodiments A1 to A21, wherein the solidphase is paramagnetic.

A23. The method of any one of embodiments A1 to A22, wherein the massspectrometry is matrix-assisted laser desorption ionization (MALDI) massspectrometry.

A24. The method of any one of embodiments A1 to A23, wherein the massspectrometry is electrospray (ES) mass spectrometry.

A25. The method of any one of embodiments A1 to A24, wherein thepresence or absence of about 1 to about 50 or more target nucleic acidsis detected.

A26. The method of any one of embodiments A1 to A25, wherein the massdistinguishable tag consists of nucleotides.

A27. The method of any one of embodiments A1 to A26, wherein the massdistinguishable tag is a nucleotide compomer.

A28. The method of embodiment A27, wherein the nucleotide compomer isabout 5 nucleotides to about 150 nucleotides in length.

A29. The method of any one of embodiments A1 to A28, wherein the targetnucleic acids are genomic DNA.

A30. The method of embodiment A29, wherein the genomic DNA is humangenomic DNA.

A31. The method of any one of embodiments A1 to A30, wherein thedetecting in (f) comprises a signal to noise ratio greater than thesignal to noise ratio for a method in which releasing does not comprisecompetition with a competitor.

B1. A method for detecting the presence, absence or amount of aplurality of genetic variants in a composition, comprising:

-   -   (a) preparing a plurality of amplicons derived from a plurality        of target nucleic acid species, or portions thereof, wherein        each target nucleic acid species comprises a first variant and a        second variant;    -   (b) hybridizing the amplicons to oligonucleotide species,        wherein each oligonucleotide species hybridizes to an amplicon        derived from a target nucleic acid species, thereby generating        hybridized oligonucleotide species; and    -   (c) contacting the hybridized oligonucleotide species with an        extension composition comprising one or more terminating        nucleotides under extension conditions; wherein:        -   (i) at least one of the one or more terminating nucleotides            comprises a capture agent, and        -   (ii) the hybridized oligonucleotide species that hybridize            to the first variant are extended by a terminating            nucleotide and the hybridized oligonucleotide species that            hybridize to the second variant are not extended by a            terminating nucleotide, thereby generating extended            oligonucleotide species;    -   (d) capturing the extended oligonucleotide species to a solid        phase that captures the capture agent;    -   (e) releasing the extended oligonucleotide species bound to the        solid phase in (d) from the solid phase; and    -   (f) detecting the mass of each extended oligonucleotide species        released from the solid phase in (e) by mass spectrometry;        whereby the presence, absence or amount of the genetic variants        is detected.

B2. The method of embodiment 1, wherein each oligonucleotide speciescomprises a mass distinguishable tag located 5′ of the hybridizationsequence

B3. The method of embodiment 1 or 2, wherein the first variant is alower abundance variation and the second variant is a higher abundancevariation.

B4. The method of any one of embodiments 1 to 3, wherein the geneticvariants are single nucleotide polymorphism (SNP) variants, the firstvariant is a lower abundance allele and the second variant is a higherabundance allele.

B5. The method of any one of embodiments 1 to 4, wherein the one or moreterminating nucleotides consist of one terminating nucleotide.

B6. The method of any one of embodiments 1 to 4, wherein the one or moreterminating nucleotides consist of two terminating nucleotides.

B7. The method of any one of embodiments 1 to 4, wherein the one or moreterminating nucleotides consist of three terminating nucleotides.

B8. The method of any one of embodiments 1 to 4, wherein the one or moreterminating nucleotides independently are selected from ddATP, ddGTP,ddCTP, ddTTP and ddUTP.

B9. The method of any one of embodiments 1 to 4 wherein the extensioncomposition comprises a non-terminating nucleotide.

B10. The method of embodiment 9, wherein the extension compositioncomprises one or more extension nucleotides, which extension nucleotidescomprise no capture agent.

B11. The method of any one of embodiments 1 to 10, wherein releasing theextended oligonucleotide species comprises contacting the solid phasewith a releasing agent.

B12. The method of embodiment 11 wherein the capture agent comprisesbiotin or a biotin analogue, the solid phase comprises streptavidin andthe releasing agent comprises free biotin or a biotin analogue.

B13. The method of embodiments 11 or 12 wherein the releasing agent hasa higher affinity for the solid phase than the capture agent.

B14. The method of any one of embodiments 11 to 13 wherein releasing theextended oligonucleotide species in (e) comprises heating from about 30°C. to about 100° C.

B15. The method of embodiment 14, comprising heating from about 60° C.to about 100° C.

B16. The method of embodiment 14, comprising heating from about 89° C.to about 100° C.

B17. The method of embodiment 14, comprising heating to about 90° C.

B18. The method of any one of embodiments 1 to 17, wherein the pluralityof target nucleic acid species is 20 or more target nucleic acidspecies.

B19. The method of any one of embodiments 1 to 18, wherein the pluralityof target nucleic acid species is 200 or more target nucleic acidspecies.

B20. The method of any one of embodiments 1 to 19, wherein the pluralityof target nucleic acid species is 200 to 300 target nucleic acidspecies.

B21. The method of any one of embodiments 1 to 20, wherein the extensionconditions in (c) comprise cycling 20 to 300 times.

B22. The method of any one of embodiments 1 to 19 wherein the extensionconditions in (c) comprise cycling 200 to 300 times.

B23. The method of any one of embodiments 1 to 22 wherein the extensionreaction comprises a competitor oligonucleotide.

B24. The method of any one of embodiments 1 to 23 comprising washing thesolid phase after the extended oligonucleotide species is captured.

B25. The embodiment of B24 wherein the washing removes salts thatproduce interfering adducts in mass spectrometry analysis.

B26. The embodiment of B25 wherein extended oligonucleotides are notcontacted with an ion exchange resin.

B27. The method of any one of embodiments B1 to B26, wherein thedetecting in (f) is with a signal to noise ratio greater than a signalto noise ratio for detecting after releasing without competition with acompetitor.

B28. The method of any one of embodiments B1 to B27, wherein a signal tonoise ratio for extending only a mutant allele is greater than a signalto noise ratio for extending a wild type and a mutant allele.

B29. The method of any one of embodiments B1 to B28, wherein thesensitivity of detecting a mutant allele in (f) is greater for extendingonly a mutant allele than for extending a wild type and a mutant allele.

B30. The method of any one of embodiments B1 to B29, wherein theextended oligonucleotide species of the second variant is not detected.

B31. The method of any one of embodiments B12 to B30, wherein the freebiotin or biotin analogue is added at a concentration from about 10 toabout 100 ug/ml.

B32. The embodiment of B31, wherein the free biotin or biotin analogueis added at a concentration of about 25 ug/ml.

B33. The method of any one of embodiments B1 to B32 wherein thecomposition comprises a synthetic template and the amount and/orpercentage of a first variant in the composition is determined whereinthe synthetic template comprises a variant different than in the firstvariant and second variant and hybridizes to the same oligonucleotidesspecies.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the technology. Although the technology has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the technology.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the technologyclaimed. The term “a” or “an” can refer to one of or a plurality of theelements it modifies (e.g., “a reagent” can mean one or more reagents)unless it is contextually clear either one of the elements or more thanone of the elements is described. The term “about” as used herein refersto a value within 10% of the underlying parameter (i.e., plus or minus10%), and use of the term “about” at the beginning of a string of valuesmodifies each of the values (i.e., “about 1, 2 and 3” is about 1, about2 and about 3). For example, a weight of “about 100 grams” can includeweights between 90 grams and 110 grams. Thus, it should be understoodthat although the present technology has been specifically disclosed byrepresentative embodiments and optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and such modifications and variations are consideredwithin the scope of this technology.

Embodiments of the technology are set forth in the claim(s) thatfollow(s).

What is claimed is:
 1. A method for detecting the presence, absence oramount of a plurality of genetic variants in a composition, comprising:(a) preparing a plurality of amplicons derived from a plurality oftarget nucleic acid species, or portions thereof, wherein each targetnucleic acid species comprises a first variant and a second variant; (b)hybridizing the amplicons to oligonucleotide species, wherein eacholigonucleotide species hybridizes to an amplicon derived from a targetnucleic acid species, thereby generating hybridized oligonucleotidespecies; and (c) contacting the hybridized oligonucleotide species withan extension composition comprising one or more terminating nucleotidesunder extension conditions; wherein: (i) at least one of the one or moreterminating nucleotide comprises a capture agent, and (ii) thehybridized oligonucleotide species that hybridize to the first variantare extended by a terminating nucleotide and the hybridizedoligonucleotide species that hybridize to the second variant are notextended by a terminating nucleotide, thereby generating extendedoligonucleotide species; (d) capturing the extended oligonucleotidespecies to a solid phase that captures the capture agent; (e) releasingthe extended oligonucleotide species bound to the solid phase in (d)from the solid phase; and (f) detecting the mass of each extendedoligonucleotide species released from the solid phase in (e) by massspectrometry; whereby the presence, absence or amount of the geneticvariants is detected.